RESUMEN

Human monocyte is an important cell type which is involved in various complex human diseases. To better understand the biology of human monocytes and facilitate further studies, we developed the first comprehensive proteome knowledge base specifically for human monocytes by integrating both in vivo and in vitro datasets. The top 2000 expressed genes from in vitro datasets and 779 genes from in vivo experiments were integrated into this study. Altogether, a total of 2237 unique monocyte-expressed genes were cataloged. Biological functions of these monocyte-expressed genes were annotated and classified via Gene Ontology (GO) analysis. Furthermore, by extracting the overlapped genes from in vivo and in vitro datasets, a core gene list including 541 unique genes was generated. Based on the core gene list, further gene-disease associations, pathway and network analyses were performed. Data analyses based on multiple bioinformatics tools produced a large body of biologically meaningful information, and revealed a number of genes such as SAMHD1, G6PD, GPD2 and ENO1, which have been reported to be related to immune response, blood biology, bone remodeling, and cancer respectively. As a unique resource, this study can serve as a reference map for future in-depth research on monocytes biology and monocyte-involved human diseases.

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Anciano , Femenino , Humanos , Persona de Mediana Edad , Biomarcadores de Tumor , Metabolismo , Proteínas de Unión al ADN , Metabolismo , Glucosafosfato Deshidrogenasa , Metabolismo , Espectrometría de Masas , Métodos , Monocitos , Metabolismo , Proteínas de Unión al GTP Monoméricas , Metabolismo , Fosfopiruvato Hidratasa , Metabolismo , Proteómica , Métodos , Proteína 1 que Contiene Dominios SAM y HD , Proteínas Supresoras de Tumor , Metabolismo

RESUMEN

OBJECTIVE: To discuss the development module of ontology-based computerized clinical practical guideline, and to supply the technology support for implement of alerm/reminding, data sharing, evidence-based medicine and clinical decision making in the medical information system. METHODS: Analyse the structure and field ontology of the Guideline Interchange Format (GLIF); expand GLIF based on the Guideline of Cerebrovascular Disease Prevention and Treatment in China and Neurology Disease Ontology. RESULTS: A Chinese computerized guideline of intravenous thrombolytic therapy of acute cerebral infarction is constructed, which included metadata of edition description, list of drug data item, explanation of evidence strength, steps of indications decision, contraindication decision and drug selection. CONCLUSION: The computerized clinical practical guideline combined with clinical information system and Electronic Medical Records plays an important role in clinical pathways optimizing and decision making.

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Toma de Decisiones Asistida por Computador , Sistemas de Apoyo a Decisiones Clínicas , Sistemas de Registros Médicos Computarizados , Guías de Práctica Clínica como Asunto , Proyectos de Investigación

RESUMEN

The Yellow Fever (YF) vaccine, an attenuated yellow fever 17D (YF-17D) live vaccine, is one of the most effective and safest vaccines in the world and is regarded as one of the best candidates for viral expression vector. We here first reported in China the construction and characterization of the recombinant expression vector of yellow fever 17D which contained the proteinase 2A fragment of foot-and-mouth disease virus (FMDV). Three cDNA fragments representing the full-length YF-17D genome, named 5'-end cDNA (A), 3'-end cDNA (B) and middle cDNA (C), were obtained by reverse transcription polymerase chain reaction (RT-PCR), together with the introduction of SP6 enhancer, necessary restriction sites and overlaps for homologous recombination in yeast. Fragment A and B were then introduced into pRS424 in turn by DNA recombination, followed by transfection of fragment C and the recombinant pRS424 containing A and B (pRS-A-B) into yeast. A recombinant vector containing full length cDNA of YF-17D (pRS-YF) was obtained by screening on medium lack of tryptophan and uracil. A recombinant YF-17D expression vector containing FMDV-2A gene fragment (pRS-YF-2A1) was then constructed by methods of DNA recombination and homologous recombination in yeast described above. In vitro transcription of the recombinant vector pRS-YF-2A1 was then carried out and introduced into BHK-21 cells by electroporation. Results of indirect immunofluorescence assay (IFA) and titer determination showed a stable infectious recombinant virus was gotten, whose features such as growth curve were similar to those of the parental YF-17D. The results suggest that the recombinant vector pRS-YF-2A1, by introduction of heterogenous genes via 2A region, is potential to be an effective live vaccine expression vector.

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Animales , Cricetinae , Línea Celular , Clonación Molecular , Epítopos , Alergia e Inmunología , Fiebre Aftosa , Virus de la Fiebre Aftosa , Genética , Alergia e Inmunología , Ingeniería Genética , Vectores Genéticos , Recombinación Genética , Saccharomyces cerevisiae , Genética , Metabolismo , Vacunas Atenuadas , Vacunas Virales , Genética , Alergia e Inmunología , Virus de la Fiebre Amarilla , Genética , Alergia e Inmunología

RESUMEN

Anti-HIV-1 gp120 single chain antibody(scFv) gene and staphylococcus extoxin A(SEA) gene were inserted into vector pPIC9K. The recombinant plasmid was integrated into Pichia pastoris by electroporation. High level expression was performed by determining the Muts phenotype and screening muti-copy integrants. The recombinant protein was about 57kD and the production was 50.1 mg/L. It was shown that the two kinds of protein affected the conformation of each other by antibody affinity assay, but the recombinant targeting toxins could highly mediate CTLs to kill HIV-1 target cells.

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Enterotoxinas , Genética , Vectores Genéticos , Proteína gp120 de Envoltorio del VIH , Genética , Alergia e Inmunología , VIH-1 , Genética , Alergia e Inmunología , Región Variable de Inmunoglobulina , Genética , Alergia e Inmunología , Pichia , Genética , Metabolismo , Proteínas Recombinantes , Genética , Alergia e Inmunología

RESUMEN

<p><b>OBJECTIVE</b>To construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6.</p><p><b>METHODS</b>The recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed.</p><p><b>RESULTS</b>There was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity.</p><p><b>CONCLUSION</b>The recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.</p>

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Animales , Embrión de Pollo , Ratones , Anticuerpos Antivirales , Sangre , Western Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Fibroblastos , Biología Celular , Metabolismo , Viruela Aviar , Sangre , Alergia e Inmunología , Virología , Virus de la Viruela de las Aves de Corral , Genética , Alergia e Inmunología , Productos del Gen gag , Genética , Metabolismo , Vectores Genéticos , Genética , Proteína gp120 de Envoltorio del VIH , Genética , Metabolismo , VIH-1 , Genética , Metabolismo , Inmunización , Métodos , Interleucina-6 , Genética , Metabolismo , Ratones Endogámicos BALB C , Microscopía Electrónica , Plásmidos , Genética , Proteínas Recombinantes de Fusión , Genética , Alergia e Inmunología , Metabolismo , Transfección , Vacunas Virales , Genética , Alergia e Inmunología , Metabolismo

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To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.

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Animales , Humanos , Ratones , Vacunas contra el SIDA , Alergia e Inmunología , Adyuvantes Inmunológicos , Anticuerpos Antivirales , Alergia e Inmunología , Virus de la Viruela de las Aves de Corral , Genética , Alergia e Inmunología , Metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Proteína gp120 de Envoltorio del VIH , Genética , Alergia e Inmunología , VIH-1 , Genética , Alergia e Inmunología , Inmunización , Interleucina-18 , Genética , Alergia e Inmunología , Ratones Endogámicos BALB C , Vacunas de ADN , Alergia e Inmunología

RESUMEN

Spider dragline silk is synthesized in special gland named major ampulate (MA) gland. The MA glands were dissected from the abdomen of the spiders Nephila clavata and the total RNA was extracted by the TRIZOL. The cDNA of dragline silk was amplificated by RT-PCR (reverse transcription polymerase chain reaction), multiplex PCR and cloned. PCR identification, restriction analysis and DNA sequence analysis were carried out to verify the recombinant plasmids. The codon usage frequencies of the cloned cDNA were added up, and the predicted amino acid sequence was compared with Spidroin2 of Nephila clavipes. Predicted secondary structure of the predicted amino-acid sequence was analysized by DNAStar software. All results showed that the cloned cDNA we got (GenBank Accession No. AF441245) was the very fragment of spider dragline silk Spidroin2 cDNA.

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Animales , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón , ADN Complementario , Química , Fibroínas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Proteínas , Química , Genética , Arañas

RESUMEN

Objective To provide experimental evidences for developing a safe and effective re- combinant fowlpox virus which can prevent the infection of HIV-2.Methods A fowlpox virus(FPV) transferring vector was constructed by inserting HIV-2 gag gene to the downstream of a synthetic complex promoter ATI-p7.5?20 of vector pUTA2.Transfection was then carried out,and recombi- nant FPV(rFPV)was screened by 5'-bromo-deoxyuridine(BrdU),genome PCR and western blot detection.Balb/c mice were immunized with rFPV by muscular injection.Anti-HIV-2 antibody, CD4~+ and CD8~+ T-cell count and specific target-killing activity of spleen CTL in immunized mice were analyzed by ELISA,FACS and LDH release assay,respectively.Results A transferring vector pA- gag was constructed and confirmed by amplifying a fragment of 766 bp from the rFPV genome.Mean- while,HIV-2 multi-antibody-specific protein blot(55 000)was detected from the recombinant virus and the HIV-2 specific antibody was detected from the immunized Balb/c mice.HIV-2 specific target- killing activity of spleen CTL was observed in immunized mice.Conclusion A recombinant fowlpox virus expressing HIV-2 structural protein Gag has been obtained,and it can stimulate HIV-2-specific eelluar and humoral immune reactions in mice.