ABSTRACT
Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.
Subject(s)
Humans , Genome, Viral , Haplotypes , Mutation , Open Reading Frames , Phylogeny , Severe acute respiratory syndrome-related coronavirus , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To fusionaly express the HAV 3a (located in 1403-1456 aa) and 3d located in 1719-1764 aa cDNA gene fragments in prokaryotic system; to investigate the antigenicity and application of recombinant protein.</p><p><b>METHODS</b>By using PCR technique, 3a and 3d gene fragments were cloned. Choosing pET-30a as the expressive vector, the recombinant plasmid Pet-3ad was constructed and pET-3ad was expressed in Escherichia coli after inducing by IPTG. By affinity chromatography, purified recombinant protein was obtained. By using Western blot analysis and indirect ELISA to detect its antigenic activity.</p><p><b>RESULTS</b>Recombinant plasmid pET-3ad was proved to construct successfully by enzyme-digestion and sequence measurement. Recombinant protein P3ad(18,000) was obtained in BL21(DE3) and purified after Ni+ affinity chromatography. Western blot analysis and indirect ELISA showed that P3ad had specific antigenicity.</p><p><b>CONCLUSIONS</b>Recombinant plasmid pET-3ad was proved to construct successfully by enzyme-digestion (Nco I/Hind III) and sequence measurement. Recombinant protein P3ad(18,000) was obtained in BL21(DE3) and purified after Ni+ affinity chromatography. Specific immunoblotting appeared at 18,000 by western blot analysis, which showed the recombinant protein P3ad had specific antigenicity, indirect ELISA further proved its antigenicity.</p>
Subject(s)
Blotting, Western , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Metabolism , Hepatitis A virus , Genetics , Plasmids , Polymerase Chain Reaction , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Viral Nonstructural Proteins , Genetics , Allergy and ImmunologyABSTRACT
<p><b>BACKGROUND</b>To get early laboratory study of type specific antigenicity of herpes simplex virus II.</p><p><b>METHODS</b>PCR and prokaryotic expression technique.</p><p><b>RESULTS</b>Herpes simplex virus II type specific gene fragment was expressed in E.coli and the products can be used as specific antigen for the detection of anti\HSV in the recovery sera.</p><p><b>CONCLUSIONS</b>Cloning and express of HSVII type specific antigen found the basis for developing specific diagnosis methods and vaccine of HSV.</p>
Subject(s)
Humans , Antigens, Viral , Allergy and Immunology , Cloning, Molecular , Gene Expression , Herpesvirus 2, Human , Genetics , Immunoglobulin G , Blood , Polymerase Chain Reaction , Recombinant Proteins , Allergy and ImmunologyABSTRACT
Objective To observe the effect of visible light on apoptosis of cultured human retinal pigment epithelium (RPE) cells. Methods Being the light source,500lx,(2 000?500)lx and (3 400?200)lx cold white light were used. The duration of exposure was 0,6,12 and 24 hours respectively. Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling, Annexin V flunorescein isothiocyanate/Propidium iodium labelling and flow cytometry. Results Apoptosis and necrosis were found in cultured human RPE cells which were exposed to visible light.(1)A significant increase in apoptotic and necrotic percentages was consistent with a higher light intensity.(2)Apoptosis was the main response to shorter (6 h and 12 h) exposure duration,while necrosis was more pronounced correlated to the prolongation of post exposure culture ( P 500 lx) increases the proportion of apoptosis and necrosis of human RPE cells in vitro.The extent is related to exposure intensity and duration. It demonstrates that the lower intensity and the shorter duration of exposure to light are, the more pronounced apoptotic percentages are observed,otherwise necrosis.
ABSTRACT
Objective To observe the effect of exogenous basic fibroblast growth factor (bFGF) on apoptosis of cultured human retinal pigment epithelial (RPE) cells exposed to visible light,and determine the role of bFGF, fibroblast growth factor receptor 1 (FGFR1),bcl 2 and caspase 3. Methods (2000? 500) lx cold white light was used. Exogenous bFGF was utilized during culture. Annexin annexin V fluorescein isothiocyanate/propidium iodium (V FITC/PI) labeling,flow cytometry, Immunocytochemical staining, enzyme associated absorb examing and reverse transcriptional polymerase chain reaction (RT PCR) were used to determine the apoptosis, the expression levels of bFGF, FGFR1, bcl 2, as well as the activity of caspase 3. Results No protective effect of bFGF was observed under the concentration 5 ng/ml. A significant inhibition of apoptosis was found in 10 ng/ml and 20 ng/ml groups ( P5 ng/ml) groups than light exposure groups ( P