RESUMO
We developed a human intracellular antibody based on the M1 protein from avian influenza virus H5N1 (A/meerkat/Shanghai/SH-1/2012) and then characterized the properties of this antibody. The M1 protein sequence was amplified by RT-PCR using the cDNA of the H5N1 virus as a template, expressed in bacterial expression system BL21 (DE3) and purified. A human strain, high affinity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian influenza virus. Further analysis demonstrated that TAT-HuScFv could inhibit the hemagglutination activity of the 300 TCID50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identified as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian influenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Aviária , Animais , Anticorpos Antivirais , China , Humanos , Proteínas Recombinantes/genéticaRESUMO
Alpha-toxin produced by Clostridium perfringens is an important virulence factor, causing food poisoning and gas gangrene in humans. As such, it is considered a potential bioterrorism threat. To date, there is still no human effective therapeutic drug against alpha-toxin. In this study, a human single chain antibody against alpha-toxin was produced from synthetic (Tomlinson I + J) naive phage display libraries, and its preventive and therapeutic efficacy in mice was examined. To prove the neutralizing potential of the scFv, alpha-toxin was preincubated with scFv and subsequently tested for its lecithinase and hemolytic activity, as well as its lethal effect in mice following intravenous administration. The equilibrium association constant between scFv and CPA was 2.02 × 1010 (1/M), as analyzed by SPR. The scFv could inhibit lecithinase and hemolytic activity, and provided effective protection against alpha-toxin when mice were challenged 1-h post scFv injection. In addition, the survival rate reached 80% for mice treated with scFv within 30 min of being challenged with a 2 × LD50 dose of alpha-toxin. These results confirmed that we successfully prepared a human scFv against C. perfringens type A alpha-toxin, which can be used in the prevention and treatment of alpha-toxin-related illness.
Assuntos
Toxinas Bacterianas/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Anticorpos de Cadeia Única/uso terapêutico , Fosfolipases Tipo C/imunologia , Animais , Toxinas Bacterianas/toxicidade , Western Blotting , Proteínas de Ligação ao Cálcio/toxicidade , Humanos , Dose Letal Mediana , Camundongos , Biblioteca de Peptídeos , Análise de Sequência de DNA , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificação , Fosfolipases Tipo C/toxicidadeRESUMO
Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy.
Assuntos
ADP Ribose Transferases/genética , Proteínas Arqueais/genética , Toxinas Bacterianas/genética , Exotoxinas/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Histonas/genética , Fatores de Virulência/genética , ADP Ribose Transferases/química , Animais , Proteínas Arqueais/química , Toxinas Bacterianas/química , Células CHO , Cricetulus , DNA Recombinante/genética , Exotoxinas/química , Terapia Genética , Células HeLa , Histonas/química , Humanos , Lipossomos , Pyrococcus horikoshii/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Transfecção/métodos , Fatores de Virulência/química , Exotoxina A de Pseudomonas aeruginosaRESUMO
The luteinizing hormone-releasing hormone- Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40), is a candidate target drug associated with elevated LHRH receptor (LHRH-R) expression in malignant tumor tissue. The capability of LHRH-PE40 to recognize LHRH-Rs on a living cell membrane was studied with single molecular recognition force spectroscopy (SMFS) based on atomic force microscopy (AFM). The recognition force of LHRH-PE40/LHRH-R was compared with that of LHRH/LHRH-R by dynamic force spectroscopy. Meanwhile, cell growth inhibition assay and fluorescence imaging were presented as complementary characterization. The results show that LHRH moiety keeps its capability to recognize LHRH-R specifically, which implies that recombinant protein LHRH-PE40 can be a promising target drug.
Assuntos
Hormônio Liberador de Gonadotropina/análogos & derivados , Análise Espectral/métodos , Hormônio Liberador de Gonadotropina/uso terapêutico , Humanos , Microscopia de Força Atômica , Neoplasias/tratamento farmacológicoRESUMO
OBJECTIVE: To detect the effect and cytotoxicity of luteinizing hormone-releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) on target cells using LHRH receptors (LHRHR). METHODS: The affinity of LHRH-PE40 and LHRH binding to LHRHR on the membrane surface of target cells were measured by enzyme linked immunosorbent assay. Morphological observations with light microscope were used to analyze its receptor pathway, with Spiegelmer, and cytotoxicity. IC(50) values of LHRH-PE40, which caused 50% inhibition of tumor cell growth were evaluated by MTT assay. The target cells were exposed to LHRH-PE40 and its cytotoxicity was analyzed by scanning and transmission electron microscopies, agarose gel electrophoresis, and flow cytometry. RESULTS: LHRH-PE40 killed target cells by LHRHR pathway. The morphological changes in these cells showed decreased cell size, cytoplasmic membrane blebbing, and chromatin condensation and margination. At a certain concentration and time point, HeLa cells were also induced to undergo programmed cell death. CONCLUSION: LHRH-PE40 induced target cells apoptosis via LHRHR.