Animals ; Cattle ; Encephalopathy, Bovine Spongiform ; epidemiology ; prevention & control ; transmission ; Humans

Objective To define laboratory technology for expression and purification of nucleocapsid proteins of human coronaviruses SARS-CoV,HCoV-229E,HCoV-OC43,HCoV-NL63 and HCoV-HKU1,animal coronaviruses bovine coronavirus,murine hepatitis virus,feline infectious peritonitis virus,and infectious bronchitis virus.MethodsThe pET-30a-based recombinant plasmids containing full length nucleocapsid(N) protein of coronaviruses were transformed into E.coil BL21(DE3) competent cells and were induced to express N proteins by IPTG.The expression products were purified by ion exchange chromatography and Ni2+ affinity chromatography,and were verified by SDS-PAGE and Western blot.Results Nine coronavirus nucleocapsid proteins with correct Mr were solubly expressed and highly purified with purity over 90%.Conclusion Successfully expressed nine recombinant nucleocapsid proteins with high purity in E.Coli,which provides materials to study the function of these coronavirus N proteins.

The development of immunotoxin DT386-GMCSF, a fusion protein which bears the N-terminal 386 amino acids of diphtheria toxin and human granulocyte-macrophage colony-stimulating factor (GM-CSF) and targets the GM-CSF receptor (GM-CSFR), has provided a promising alternative therapy to the acute myeloid leukemia (AML). However, the poor expression of the protein in E.coli is still a bottleneck which limits the industrial production. To identify the critical down-regulating factors on the expression of DT386-GMCSF, a series of truncated mutants of DT386-GMCSF at the C-terminal of GM-CSF were generated and expressed in E.coli. The results showed that the encoding sequences for the L114 of the GM-CSF dramatically impact the expression of DT386-GMCSF. On this basis, a serial of mutants integrating amino acid substitutes were generated. The results revealed that the expression level of the mutant DF123GVT, which harbors the amino acids 1-123 of GM-CSF whose L114L115V116 was substituted with G114V115T116, was evidently higher than that of the DT386-GMCSF, whereas the specific cytotoxicity to blast recovered from mice injected with HL60, a cell line highly expresses the GM-CSFR, was similar. These results have provided an important basis for the future development of the immunotoxins targeting the GM-CSFR.

Hantaan virus ; genetics ; isolation & purification ; Hemorrhagic Fever with Renal Syndrome ; prevention & control ; virology ; Humans ; Viral Vaccines ; immunology

OBJECTIVETo establish a method that can quantitatively detect S100 protein in CSF, and evaluate the possibility in diagnosis of Creutzfeldt-Jakob disease (CJD).

METHODSS100 gene was amplified by PCR from a commercially supplied human brain cDNA library. After verified by sequence analysis, the full length of S100 DNA was subcloned into a (GST) expression vector Pgex-2T, and the expression of GST-S100 fusion protein was induced. Rabbits were immunized with the purified GST-S100 fusion protein, and the antiserum raised against S100 protein was collected and further evaluated. Using biotin-avidin system, a sandwich ELISA was established for quantitatively determining S100 protein, and further, used in screening for S100 protein in CSF and serum samples.

RESULTSSDS-PAGE assays yielded a roughly 35,000 GST-S100 fusion protein. Using the established method, three CSF samples from probable CJD patients (14-3-3 protein positive in CSF) showed higher concentration of S100 protein (higher than 2.900 microg/L), whereas other CSF samples collected from patients with other CNS diseases showed lower concentration of S100 (less than 0.180 microg/L).Moreover, the sera S100 proteins from all the collected samples showed distinct individual difference.

CONCLUSIONSThe established method can be used in determining S100 protein in CSF quantitatively. The feasibility and significance of S100 protein in CSF for diagnosis of CJD should be further considered with more CSF samples.

Animals ; Creutzfeldt-Jakob Syndrome ; cerebrospinal fluid ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immune Sera ; biosynthesis ; Rabbits ; S100 Proteins ; biosynthesis ; cerebrospinal fluid ; Sensitivity and Specificity

BACKGROUNDTo investigate the prevalence of the parvovirus B19 infection among the blood donors in Jilin province to provide the basic data to evaluate the epidemics of B19 virus in China.

METHODSIndirect ELISA was used to detect IgG antibody against parvovirus B19 in the sera from blood donors.

RESULTSIn a total of 184 serum samples, IgG antibody was detected in 55.43% samples, antibody positive rate in female was significantly higher than that in male (P<0.05) and the positive rate peaked at 35-45 years age group.

CONCLUSIONThese data illustrate that the prevalence of the B19 antibody in blood donors of Jilin province was high, and it is therefore necessary to detect the B19 DNA to ensure the blood safety.

Adolescent ; Adult ; Antibodies, Viral ; blood ; Blood Donors ; China ; epidemiology ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Humans ; Immunoglobulin G ; blood ; Male ; Middle Aged ; Parvoviridae Infections ; blood ; epidemiology ; immunology ; Parvovirus B19, Human ; immunology ; Young Adult

BACKGROUNDTo clarify the features of gene variation among epidemic strains of human rotavirus NSP4 in China.

METHODSSP4 genes from 27 epidemic strains of human rotavirus isolated in different area of China in recent years were amplified with RT-PCR, the resulted cDNAs were cloned and sequenced. The sequences of full length cDNAs were compared with 10 rotavirus NSP4 sequences available in the GenBank using the Clustal x 1.8 TreeView32 and DNA Star softwares. The G serotype of VP7 was analysed by PCR.

RESULTSThe homology of the amino acid among the 27 rotavirus strains isolated in China was 81.7%-99.4%. Based on the variation of amino acid sequence, the virus strains can be divided into two groups, represented by Wa and KUN with the homology of 92.0%-99.4% and 92.0%-98.9% within each group, respectively. The diversity between the two groups were 16.6%-21.0%. The Wa group could further be separated into three subgroups, according to the diversity between those strains and the characterization in the highly variable domain. The association between VP7 serotype and NSP4 genotype was not strong.

CONCLUSIONSThe NSP4 gene of human rotavirus epidemic strains in China can be divided into Wa and KUN two groups, Wa group is the main group and contains three subgroups possessing characteristic amino acid sites. Samples isolated in the same year but not in the same area shared higher homology.

Antigens, Viral ; Capsid Proteins ; genetics ; DNA, Complementary ; analysis ; DNA, Viral ; analysis ; Diarrhea ; virology ; Genetic Variation ; Genotype ; Glycoproteins ; genetics ; Humans ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Rotavirus ; genetics ; isolation & purification ; Sequence Analysis, DNA ; Toxins, Biological ; Viral Nonstructural Proteins ; genetics

AIMTo investigate the possible role of manganese in the regulation of mitochondrial aconitase (mACON) activity human prostate carcinoma cell line PC-3 cells.

METHODSThe mACON enzymatic activities of human prostate carcinoma cell line PC-3 cells were determined using a reduced nicotinamide adenine dinucleotide-coupled assay. Immunoblot and transient gene expression assays were used to study gene expression of the mACON. The putative response element for gene expression was identified using reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays.

RESULTSIn vitro study revealed that manganese chloride (MnCl2) treatment for 16 h inhibited the enzymatic activity of mACON, which induced the inhibition of citrate utility and cell proliferation of PC-3 cells. Although results from transient gene expression assays showed that MnCl2 treatment upregulated gene translation by approximately 5-fold through the iron response element pathway, immunoblot and reporter assays showed that MnCl2 treatments inhibited protein and gene expression of mACON. This effect was reversed by co-treatment with ferric ammonium citrate. Additional reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays suggested that a putative metal response element in the promoter of the mACON gene was involved in the regulation of MnCl2 on the gene expression of mACON.

CONCLUSIONThese findings suggest that manganese acts as an antagonist of iron, disrupting the enzymatic activity and gene expression of mACON and citrate metabolism in the prostate.

Aconitate Hydratase ; antagonists & inhibitors ; genetics ; Actins ; genetics ; Adenosine Triphosphate ; metabolism ; Cell Line, Tumor ; Chlorides ; pharmacology ; Citrates ; metabolism ; DNA Primers ; Gene Expression Regulation, Enzymologic ; drug effects ; Gene Expression Regulation, Neoplastic ; drug effects ; Genes, Reporter ; Humans ; Iron ; metabolism ; Male ; Manganese Compounds ; pharmacology ; Mitochondria ; enzymology ; Prostatic Neoplasms ; enzymology

OBJECTIVETo investigate the genetic stability during the serial passages of non-replicating recombinant adenoviruses based on the novel AdEasy system.

METHODSFour non-replicating adenoviruses expressing rotavirus antigens, which were generated by the AdEasy system, were used as models and continuously propagated on 293 cells for 20 passages. Samples of the infected cells were collected at every 5 passages for the PCR analysis of the inserted rotavirus genes, replication-competent adenoviruses (RCAs) as well as the Western blot examination of the expression of the certain rotavirus genes.

RESULTSThe adenoviruses can be stably propagated on 293 cells. The existence and the stable expression of inserted rotavirus genes were able to be detected during the serial passage. The RCAs were not found within the first 10 passages, but the rvAdG2VP7(o) at passage 15 and the rvAdG2VP7(o) and rvAdG1VP7(o) at passage 20.

CONCLUSIONThe non-replicating adenoviruses showed promising genetic stability during the continuous passage on 293 cells. The RCAs normally will not be detectable within 10 continuous passages. The results indicated the potential of such recombinant adenoviruses in the research and development of the gene therapies and adenoviral-vectored vaccines.

Adenoviridae ; genetics ; isolation & purification ; physiology ; Cell Line ; DNA, Recombinant ; genetics ; Humans ; Serial Passage ; Transgenes ; Virus Replication

OBJECTIVETo obtain monoclonal antibodies (McAbs) which can be widely used to detect mammalian prions (PrP) and to develop diagnostic tests for screening transmissile spongiform encephalopathies (TSE) as well as for studying pathogenesis of prion-related diseases.

METHODSBALB/c mice were immunized separately with bovine PrP peptide 29-48 (BoP1) and 89-108 (BoP2) coupled to keyhole limpt hemocyan. Two hybridoma cell lines secreting monoclonal antibodies against these peptides were established by cell fusion and 2 to 3 rounds of cell cloning. The reactions of the McAbs to the recombinant bovine (Bo)PrP(25-242), human (Hu)PrP(23-231) and hamster (Ha) PrP (23?231) were tested separately by Western blotting.

RESULTSThrough cell fusion, two hybridoma cell lines secreting McAbs against BoP1 and BoP2, designated D11 and D8 accordingly, were identified by ELISA and cell cloning. The McAbs produced by these cell lines reacted well with the recombinant PrP proteins; (Bo) PrP (25-242), (Hu) PrP (23-231), and (Ha) PrP (23-231), respectively.

CONCLUSIONSTwo McAbs reacting with bovine, human and hamster PrPs were successfully generated, they are potential to be used to detect PrPs in mammals and to study the mechanism of pathogenesis of TSE.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibodies, Viral ; biosynthesis ; immunology ; Antibody Specificity ; Cattle ; Cricetinae ; Cross Reactions ; Encephalopathy, Bovine Spongiform ; prevention & control ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Hybridomas ; secretion ; Male ; Mice ; Mice, Inbred BALB C ; PrPSc Proteins ; immunology ; Prion Diseases ; prevention & control ; Prions ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology