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1.
Acta Physiologica Sinica ; (6): 301-308, 2022.
Article in Chinese | WPRIM | ID: wpr-927606

ABSTRACT

Nogo-B receptor (NgBR) is a specific receptor of Nogo-B, a member of reticulon 4 protein family. It is widely expressed in many tissues and mainly located in cell membrane and endoplasmic reticulum. Previous studies have revealed that NgBR is involved in a variety of physiological and pathophysiological processes, such as dolichol synthesis, lipid metabolism, cholesterol trafficking, insulin resistance, vascular remodeling and angiogenesis, tumorigenesis and nervous system diseases. Further studies on the molecular characteristics and biological function of NgBR might be of great significance to understand its role in diverse diseases and provide possible clinical strategies for the treatment of diseases.


Subject(s)
Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Lipid Metabolism , Nogo Proteins/metabolism , Receptors, Cell Surface/metabolism
2.
Chinese Medical Journal ; (24): 2237-2241, 2019.
Article in English | WPRIM | ID: wpr-802933

ABSTRACT

Objective@#Chronic rhinosinusitis (CRS) involves inflammation of the nasal and para-nasal mucosa. Due to its heterogeneous nature, unknown pathogenesis, and high recurrence rate, effective treatment is difficult. Nasal cytology is presently not a part of the routine diagnosis or treatment decision for CRS.@*Data sources@#A literature search was performed for published papers in English between January 1990 and June 2019 using MEDLINE.@*Study selection@#Terms used were chronic rhinosinusitis, eosinophils, etiology, immunopathology, inflammation, mast cells, nasal cytology, polyps, and treatment. Both reviews and original articles were collected and studied.@*Results@#There is no standard nasal fluid, mucus sampling, or staining techniques for identifying inflammatory cell types. Results were divergent from different countries. Moreover, the main focus of these papers on the cells in nasal washings was eosinophils, with infrequent mentioning of other cell types that may imply different etiology and pathology. The heterogeneous cell profile of CRS and the role of mast cells have been unappreciated due to the lack of specific immunohistochemical technique or study of its unique mediators.@*Conclusions@#Nasal cytology could help distinguish the type and the activation state of inflammatory cells. Thus it can help in providing a clearer picture of CRS pathogenesis, identifying different patient groups, and developing effective treatments.

3.
Chinese Medical Journal ; (24): 2237-2241, 2019.
Article in English | WPRIM | ID: wpr-774667

ABSTRACT

OBJECTIVE@#Chronic rhinosinusitis (CRS) involves inflammation of the nasal and para-nasal mucosa. Due to its heterogeneous nature, unknown pathogenesis, and high recurrence rate, effective treatment is difficult. Nasal cytology is presently not a part of the routine diagnosis or treatment decision for CRS.@*DATA SOURCES@#A literature search was performed for published papers in English between January 1990 and June 2019 using MEDLINE.@*STUDY SELECTION@#Terms used were chronic rhinosinusitis, eosinophils, etiology, immunopathology, inflammation, mast cells, nasal cytology, polyps, and treatment. Both reviews and original articles were collected and studied.@*RESULTS@#There is no standard nasal fluid, mucus sampling, or staining techniques for identifying inflammatory cell types. Results were divergent from different countries. Moreover, the main focus of these papers on the cells in nasal washings was eosinophils, with infrequent mentioning of other cell types that may imply different etiology and pathology. The heterogeneous cell profile of CRS and the role of mast cells have been unappreciated due to the lack of specific immunohistochemical technique or study of its unique mediators.@*CONCLUSIONS@#Nasal cytology could help distinguish the type and the activation state of inflammatory cells. Thus it can help in providing a clearer picture of CRS pathogenesis, identifying different patient groups, and developing effective treatments.

4.
Chinese Journal of Virology ; (6): 599-604, 2014.
Article in Chinese | WPRIM | ID: wpr-280321

ABSTRACT

To establish a MacELISA method for the detection of IgM antibodies against Chikungunya virus (CHIKV), we prepared virus like particle (VLP) antigens of CHIKV using the whole structural protein C-E3-E2-6K-E1 encoding gene with a baculovirus expression system in Sf9 insect cells. The VLPs were purified and used to immunize Kunming mice. Then, polyclonal antibodies were purified from the samples of ascites with a protein G HiTrap SP column and labeled with horseradish peroxidase. A MacELISA method for the detection of IgM antibodies against CHIKV was assembled with goat anti-human IgM antibody, VLP antigens and an enzyme-labeled polyclonal antibody. The results were evaluated with a serum panel containing serum samples from laboratory-confirmed CHIK, HFRS patients, healthy donors, and commercially available CHIKV IgM as a quality control. It was shown that the MacELISA had a specificity of 99% (99/100), the coefficients of variation (CoV) within a plate were <10%, and the CoV of different ELISA plates in terms of the plate variation coefficient was <15%. A comparative analysis was performed to compare the current method against a commercial CHIKV IgM antibody detection kit for IIFA-IgM. The detection limit of MacELISA was significantly lower than that of the IIFA-IgM commercial kit (P< 0.0001). Here, we demonstrate that the VLP-based MacELISA is a promising tool for the early diagnosis and epidemiological investigation of CHIKV infection, with a high level of sensitivity and specificity for the detection of IgM antibodies against CHIKV.


Subject(s)
Animals , Humans , Mice , Antibodies, Viral , Blood , Chikungunya Fever , Blood , Diagnosis , Virology , Chikungunya virus , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Methods , Immunoglobulin M , Blood
5.
Chinese Journal of Virology ; (6): 432-436, 2013.
Article in Chinese | WPRIM | ID: wpr-339932

ABSTRACT

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.


Subject(s)
Humans , Bunyaviridae Infections , Diagnosis , Virology , Cell Line , DNA Ligases , Metabolism , DNA, Complementary , Genetics , DNA-Directed DNA Polymerase , Metabolism , Dengue , Diagnosis , Virology , Dengue Virus , Genetics , Genome, Viral , Genetics , Phlebovirus , Genetics , RNA, Viral , Genetics , Reference Standards , Reverse Transcriptase Polymerase Chain Reaction , Methods , Viral Load
6.
Chinese Journal of Experimental and Clinical Virology ; (6): 215-217, 2013.
Article in Chinese | WPRIM | ID: wpr-318060

ABSTRACT

<p><b>OBJECTIVE</b>To develop an assay for titration of severe fever with thrombocytopenia syndrome virus (SFTSV) based on double antibody sandwich ELISA.</p><p><b>METHODS</b>A double antibody sandwich ELISA was developed for detection of SFTSV based on SFTSV nucleocapsid (N) protein specific poly- and monoclonal antibodies, procedures were optimized and evaluated. This ELISA based titration assay was compared with fluorescence assasy and plaque assay based titration method.</p><p><b>RESULTS</b>The results suggested that the titers obtained by ELISA based method are consistent with those obtained by IFA based method (R = 0.999) and the plaque assay titration method (R = 0.949).</p><p><b>CONCLUSION</b>The novel ELISA based titration method with high sensitivity and specificity is easy to manage and perform, and can overcome the subjectivity associated with result determination of the fluorescence assay and plaque assay based methods. The novel ELISA based titration method can also be applied to high throughput detection.</p>


Subject(s)
Humans , Bunyaviridae , Enzyme-Linked Immunosorbent Assay , Methods , Fever , Virology , Fluorescent Antibody Technique , Thrombocytopenia , Virology
7.
Chinese Journal of Experimental and Clinical Virology ; (6): 257-259, 2013.
Article in Chinese | WPRIM | ID: wpr-318049

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the data of surveillance on severe fever with thrombocytopenia syndrome (SFTS), from 2011 to 2012 in China.</p><p><b>METHODS</b>Descriptive methods were conducted to analyze the surveillance data from 2011 to 2012 which were collected from the internet-based National Notifiable Disease Reporting System.</p><p><b>RESULTS</b>From 2011 to 2012, a total of 1229 SFTS cases and 107 deaths were reported in China with the average annual incidence rate of 0. 046/100 000 and case fatality rate of 8.7%. Compared to 2011, morbidity of 2012 has increased by 23.5% and mortality has decreased by 32%. 16 provinces reported SFTS cases. More cases occurred in spring and summer seasons,with the peak in May to July, during this period, 69% of the total cases were reported. The ages of the patients ranged from 1 to 85 years, 44.2% of total case was 55 to 70 years old, there were no differences in sex. Of all the cases 86. 8% was farmer.</p><p><b>CONCLUSION</b>Severe fever with thrombocytopenia syndrome in widely distributed in China, especially in the central and eastern regions, the incidence has obvious seasonal. Surveillance and immigration quarantine should be strengthened.</p>


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Humans , Infant , Male , Middle Aged , Young Adult , China , Epidemiology , Epidemics , Phlebotomus Fever , Epidemiology , Mortality , Virology , Phlebovirus , Classification , Genetics , Sentinel Surveillance
8.
Chinese Journal of Experimental and Clinical Virology ; (6): 456-459, 2012.
Article in Chinese | WPRIM | ID: wpr-305010

ABSTRACT

<p><b>OBJECTIVE</b>A 12 mer phage display peptide library was used to identify hepatitis A virus mimotopes of antigenic determinants, to provide the feasibility of virus epitope mapping by using this approach.</p><p><b>METHODS</b>Using purified anti-hepatitis A virus monoclonal antibody as affinity selective molecule, phage display peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing.</p><p><b>RESULTS</b>10 ELISA positive clones were chosen for DNA sequencing, and the displayed peptide sequences were deduced. 9 of them showed identical nucleotide sequence, and similarity in their amino acid sequence with VP1 of HAV HM175 was found, but no sequence homology was found between the other phage clone and the capsid proteins of HAV. Those peptides may behave as mimotopes of HAV.</p><p><b>CONCLUSION</b>The mimotope of HAV was selected by using phage display peptide library screening. The results provide the potential of this method to search for the mimotopes of the virus.</p>


Subject(s)
Humans , Amino Acid Sequence , Antigens, Viral , Chemistry , Genetics , Allergy and Immunology , Epitope Mapping , Epitopes , Hepatitis A , Virology , Hepatitis A virus , Chemistry , Genetics , Allergy and Immunology , Molecular Sequence Data , Peptide Library
9.
Chinese Journal of Otorhinolaryngology Head and Neck Surgery ; (12): 317-319, 2012.
Article in Chinese | WPRIM | ID: wpr-313575

ABSTRACT

<p><b>OBJECTIVE</b>To verify the correlation between nasal contact point and headache and to discuss the significance of nasal endoscopic surgery through observation of clinical outcomes in patients with nasal mucosa contact point treated by endoscopic surgery.</p><p><b>METHODS</b>Forty-five patients diagnosed as nasal mucosa contact headache were treated by nasal endoscopic surgery. The results were analyzed retrospectively, including headache degree, headache frequency, lasting time and total time between before and 6, 18, and 24 months after operation. The data were processed by rank-sum test by SPSS 18.0 software.</p><p><b>RESULTS</b>6, 18 and 24 months after operation, the headache degree (VAS scores were 1.50 [0; 4.00], 2.00 [0; 5.00], 3.25 [0; 5.75], respectively) was relieved (VAS score was 6.00 [5.25; 8.25] before operation) dramatically (Z value were -4.913, -4.070 and -3.095, respectively, all P < 0.01). The total time of headache 6, 18, 24 months after operation (It were 0.07 [0; 3.50], 0.26 [0; 7.77], 1.04[0; 17.15] h, respectively) was shortened (It was 25.20 [4.00; 186.00] h before operation) significantly (Z value were -4.368, -3.652, -2.500, respectively, all P < 0.05).</p><p><b>CONCLUSIONS</b>One of the key causes of patients suffered from intractable headaches is mucosal contact in the nasal cavity. The pain in these patients could be relieved through surgical correction of intranasal anatomic abnormalities. Nasal mucosa contact might not be the only etiology of intractable headache since the mechanism of headache is complicated and variable. The effect of endoscopic surgery needs to be estimated by long-term follow-up.</p>


Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Headache , Pathology , Nasal Mucosa , Pathology , Pain Measurement , Retrospective Studies , Turbinates
10.
Chinese Journal of Virology ; (6): 252-257, 2012.
Article in Chinese | WPRIM | ID: wpr-354739

ABSTRACT

To understand the maintenance and transmission of SFTS virus, the potential vector ticks were collected from sheep, cattle and dogs in the endemic areas of SFTSV in Shandong Province. Among the collected ticks, the dominant species was H. longicornis ticks. Real-time PCR for RNA detection, virus isolation and characterization, genomic sequencing, phylogenetic and antigenic analysis were performed in this investigation. The results showed that the SFTS viral RNA was detected in 2.14% H. longicornis, and a SFTS virus was isolated from one of viral RNA positive ticks collected from sheep. Whole genome analysis of the SFTSV isolates with 11 human-origin SFTS virus revealed a highly pairwise similarity, and the growth curve analysis showed nearly identical in virus yield and the dynamic of virus reproduction compared to human derived viral isolates. Immunofluorescence and neutralization test showed identical serological reaction character of the two different origin viral strains. In this study, the characters of a SFTSV isolate was firstly described, which suggested that the tick species H. longicornis acting important vector role in the transmission of SFTS virus.


Subject(s)
Animals , Cattle , Dogs , Humans , Animals, Domestic , Parasitology , Arachnid Vectors , Virology , Bunyaviridae Infections , Virology , Cell Line , Livestock , Parasitology , Molecular Sequence Data , Phlebovirus , Classification , Genetics , Phylogeny , Sheep , Ticks , Virology
11.
Chinese Journal of Cardiology ; (12): 390-396, 2011.
Article in Chinese | WPRIM | ID: wpr-272236

ABSTRACT

<p><b>OBJECTIVE</b>To observe the clinical characteristics, treatment options and outcome of diabetic patients with non-ST elevation acute coronary syndromes (NSTEACS).</p><p><b>METHODS</b>Consecutive patients admitted with NSTEACS from 38 centers in north China were enrolled. Medical histories, clinical characteristics, treatments and outcomes were evaluated and follow-up was made at 6, 12, and 24 months after their initial hospital admission. Cumulative event rates were compared between diabetic and non-diabetic patients.</p><p><b>RESULTS</b>There were 420 diabetic patients out of 2294 NSTEACS patients (18.3%). Diabetic patients were older [(64.9 ± 6.7) years vs. (62.3 ± 8.6) years, P < 0.01], more often women (48.1% vs. 35.3%, P < 0.05) and were associated with higher baseline comorbidities such as previous hypertension, myocardial infarction, congestive heart failure and stroke than non-diabetic patients. The incidence of antiplatelet therapy (92.1% vs. 95.0%, P < 0.05), coronary angiography (30.0% vs. 36.3%, P < 0.05) and revascularization (12.1% vs.18.8%, P < 0.05) was lower in patients with diabetes than non-diabetic patients. In hospital and 2-year mortality as well as the incidence of congestive heart failure and composite outcomes of myocardial infarction, stroke, congestive heart failure and death were substantially higher in diabetic patients compared with non-diabetic patients. Multivariate Cox regression analysis revealed that age ≥ 70 years, diabetes, previous myocardial infarction, previous congestive heart failure, systolic blood pressure less than 90 mm Hg (1 mm Hg = 0.133 kPa) and heart rate more than 100 bpm at admission were risk factors for 2-year death.</p><p><b>CONCLUSION</b>In NSTEACS, diabetes is associated with higher rate of in-hospital and 2-year death, congestive heart failure and composite outcomes of myocardial infarction, stroke, congestive heart failure and death. Diabetes mellitus is a major independent predictor of 2-year mortality post NSTEACS. Status of antiplatelet therapy, coronary angiography and revascularization should be improved for diabetic patients with NSTEACS during hospitalization.</p>


Subject(s)
Aged , Female , Humans , Male , Middle Aged , Acute Coronary Syndrome , Diagnosis , Epidemiology , Therapeutics , China , Epidemiology , Diabetes Complications , Diagnosis , Epidemiology , Therapeutics , Electrocardiography , Follow-Up Studies , Prognosis , Regression Analysis , Treatment Outcome
12.
Chinese Journal of Experimental and Clinical Virology ; (6): 85-88, 2011.
Article in Chinese | WPRIM | ID: wpr-231186

ABSTRACT

<p><b>OBJECTIVE</b>To secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly.</p><p><b>METHODS</b>The entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, SA14-14-2 strain) was introduced using fusion PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3' terminus and one chimeric gene, which was generated by replacing the 3' terminal 20% region of E gene with the corresponding sequence of JEV (SA14-14-2 strain). The PCR segments were inserted into the NheI and NotI sites of pcDNA5/FRT vector or into the NheI and XhoI sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay (IFA) and Western Blot.</p><p><b>RESULTS</b>After transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis.</p><p><b>CONCLUSION</b>Signal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.</p>


Subject(s)
Animals , Humans , Cell Line , Dengue , Metabolism , Virology , Dengue Virus , Genetics , Metabolism , Gene Expression , Protein Structure, Tertiary , Protein Transport , Spodoptera , Viral Envelope Proteins , Chemistry , Genetics , Metabolism
13.
Journal of Southern Medical University ; (12): 665-668, 2011.
Article in Chinese | WPRIM | ID: wpr-332579

ABSTRACT

<p><b>OBJECTIVE</b>To observe the inhibitory effect of resveratrol against the cytopathogenicity of enterovirus type 71.</p><p><b>METHODS</b>The cytotoxicity of resveratrol on Vero cells was detected using cell counting kit-8 (CCK-8). The antiviral activity of resveratrol in different stages of infection, with ribavirin as the control, was evaluated by determining the virus inhibition rate, medium effective concentration (IC(50)), and selection index (SI).</p><p><b>RESULTS</b>Resveratrol was nonpoisonous to Vero cells with an median toxic concentration (TC50) of 307.6 mmol/L. Resveratrol produced an obvious inhibitory effect against enterovirus type 71 only before the cell infection by the virus (IC(50)=20.2 mmol/L , SI=15.2), and once the cells were infected, resveratrol no longer had such antiviral effect.</p><p><b>CONCLUSIONS</b>Resveratrol may offer some protection against enterovirus type 71 in vitro.</p>


Subject(s)
Animals , Antiviral Agents , Pharmacology , Chlorocebus aethiops , Drugs, Chinese Herbal , Pharmacology , Enterovirus , Stilbenes , Pharmacology , Vero Cells
14.
Chinese Journal of Cardiology ; (12): 1065-1072, 2010.
Article in Chinese | WPRIM | ID: wpr-244104

ABSTRACT

<p><b>OBJECTIVE</b>To compare the impact of the first 24 hours mean blood glucose (MBG) level and admission glucose (AG) during hospitalization on the short term mortality and combined end point events in patients with ST-segment elevation acute myocardial infarction (STEMI).</p><p><b>METHODS</b>A total of 7446 Chinese STEMI patients hospitalized within 12 hours of symptom onset were included. Plasma glucose was measured at admission, 6 and 24 hours after admission, respectively. The MBG level through the first 24 hours for each patient was calculated. Patients were stratified into six groups according to their MBG levels: < 4.5, 4.5 - 5.5, 5.6 - 7.0, 7.1 - 8.5, 8.6 - 11.0 and > 11.0 mmol/L. The incidence of all-cause mortality and combined end point of death, re-infarction, cardiogenic shock, recurrence ischemia, and stroke at 7 days and 30 days post hospitalization were analyzed. Nested models were compared to determine whether logistic regression models that included MBG provided a significantly better fit than logistic regression models included AG.</p><p><b>RESULTS</b>Compared with the MBG of 4.5 - 5.5 mmol/L group, 7-day and 30-day mortality and combined end point events increased in proportion to plasma MBG level increase. Multivariate logistic regression analysis showed that elevated MBG (equal or greater than 7.1 - 8.5 mmol/L) level is an independent predictor of 7-day and 30-day mortality and combined end point events. Nested models analysis showed that the prognostic impact of MBG is superior to AG (P < 0.001) on predicting 7-day and 30-day mortality and combined end point events in this patient cohort.</p><p><b>CONCLUSION</b>Elevated MBG (≥ 7.1 mmol/L) level is an independent predictor of 7-day and 30-day mortality and combined end point events. MBG is superior to AG on predicting short-term prognosis in this patient cohort.</p>


Subject(s)
Aged , Female , Humans , Male , Middle Aged , Blood Glucose , China , Electrocardiography , Endpoint Determination , Hospital Mortality , Logistic Models , Multivariate Analysis , Myocardial Infarction , Diagnosis , Mortality , Prognosis
15.
Chinese Journal of Experimental and Clinical Virology ; (6): 56-58, 2010.
Article in Chinese | WPRIM | ID: wpr-316966

ABSTRACT

<p><b>OBJECTIVE</b>Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus.</p><p><b>METHODS</b>The internal controls target gene were obtained by insertion of a 180 bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions.</p><p><b>RESULTS</b>The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis.</p><p><b>CONCLUSION</b>The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.</p>


Subject(s)
Humans , Cell Line , DNA, Viral , Genetics , Dengue , Diagnosis , Virology , Dengue Virus , Genetics , Reference Standards , Reverse Transcriptase Polymerase Chain Reaction , Methods , Reference Standards
16.
Chinese Journal of Virology ; (6): 185-189, 2009.
Article in Chinese | WPRIM | ID: wpr-334753

ABSTRACT

The prM/E gene of DV2 was cloned into the JEV (SA14-14-2 strain) replicon vector which had been constructed previously, and the resulting recombinant plasmid was named pPartialdeltaprM/E. The constructed chimeric clone was linearized and then was transcripted into RNA in vitro. The produced RNA was transfected into BHK-21 cells. Five to seven days later, CPE could be observed on the transfected BHK-21cells, and then the supernatant containing the chimeric virus was collected. The Supernatant was inoculated to BHK-1 cells and C6/36 cells, respectively. CPE could be observed about 4 days post the infection of C6/36cell with the chimeric virus. The results from RT-PCR, IFA, Western blot showed that the virus contained the chimeric RNA and the envelop protein of DV2. However, the chimeric virus could not be passaged in BHK-21 cell. The successful construction of the infectious clone JE/DEN-2 laid the basis for the further research of the DV vaccine.


Subject(s)
Animals , Cricetinae , Blotting, Western , Cell Line , Dengue Virus , Genetics , Encephalitis Viruses, Japanese , Genetics , Genetic Vectors , Genetics , Reassortant Viruses , Genetics , Recombination, Genetic , Genetics , Reverse Transcriptase Polymerase Chain Reaction
17.
Chinese Journal of Experimental and Clinical Virology ; (6): 149-151, 2009.
Article in Chinese | WPRIM | ID: wpr-332399

ABSTRACT

<p><b>OBJECTIVE</b>To develop and evaluate a multiplex detection of IgM antibodies to pathogens caused viral hemorrhagic fever.</p><p><b>METHODS</b>The nucleocapsid proteins (NP) of HTN, SEO, Puu MBV, Lassa, RFV and HPS viruses expressed in prokaryotic cells and purified were coupled to 7 different xMAP fluorescent microbeads. The assay was evaluated and optimized when screened against a panel of reference sera collected from HFRS patients, and compared to commonly used MacELISA Kits.</p><p><b>RESULTS</b>For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commonly used MacELISA kit, but it could detect different antigen specific antibodies in one reaction simultaneously.</p><p><b>CONCLUSION</b>A robust, rapid and multiplex assay based on NPs could be developed via Luminex xMAP platform for laboratory diagnosis of viral hemorrhagic fever and seroepidemiological investigation.</p>


Subject(s)
Humans , Antibodies, Viral , Blood , Fluorescence , Hemorrhagic Fevers, Viral , Blood , Allergy and Immunology , Virology , Immunoassay , Methods , Immunoglobulin M , Blood , Microspheres , Nucleocapsid Proteins , Chemistry , Allergy and Immunology , Viruses , Allergy and Immunology
18.
Chinese Journal of Experimental and Clinical Virology ; (6): 415-417, 2009.
Article in Chinese | WPRIM | ID: wpr-325526

ABSTRACT

<p><b>OBJECTIVE</b>To expression prM/E gene of dengue virus type I in mammalia cells.</p><p><b>METHODS</b>The full-length prM/E gene of dengue virus type I strain GZ01/95 was amplified by RT-PCR, the signal peptide preceding the prM gene was added or the carboxyl-terminal 20% of DEN-1 E was replaced with the corresponding JE sequence in the meanwhile, and three of the constructions were cloned into the pcDNA5/FRT.Then they were transfected into 293T cells by lipofectamine respectively. The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA) as well as Western blot.</p><p><b>RESULTS</b>In the cytoplasm of 293T cells transfected with all the recombinant plasmids DNA, the expressed products for gene of dengue virus type I were confirmed by IFA. The secreted expression products for gene of dengue virus type I specific protein bands were confirmed by Western blot only existing in the cell supernatants transfected with the modified recombinant plasmids DNA.</p><p><b>CONCLUSION</b>The prM/E protein of dengue virus type 1 were expressed in 293T cells transfected with all the three recombinant plasmids DNA. The prM/E protein was obtained secretion after transfecting the modified recombinant plasmids adding a signal peptide preceding the prM gene or replacing the carboxyl-terminal 20% of E with the corresponding JE sequence.</p>


Subject(s)
Humans , Cell Line , Dengue , Virology , Dengue Virus , Genetics , Metabolism , Gene Expression , Glycoproteins , Genetics , Metabolism , Protein Transport , Recombinant Fusion Proteins , Genetics , Metabolism , Viral Envelope Proteins , Genetics , Metabolism
19.
Chinese Journal of Experimental and Clinical Virology ; (6): 418-420, 2009.
Article in Chinese | WPRIM | ID: wpr-325525

ABSTRACT

<p><b>OBJECTIVE</b>In order to lay the groundwork for studying the novel vaccine Identified.</p><p><b>METHODS</b>(1) Two replicons were constructed. One's prM/E gene was deleted completely (Full AprM/E Replicon), the other's prM/E gene was deleted partially (213 bp of C terminal of E gene was reserved; Partial delta prM/E Replicon), and the deleted parts was replaced as the MCS. (2) Replicons RNA were which will use the JEV as the vector, replicon vectors of JEV was constructed and transfected into BHK-21 cell. After 24, 48, 72, 96 h, method of real-time PCR was used to identify Replicons' replication ability. (3) YFP gene was inserted into the MCS of those two replicons. Their RNA was transfected into BHK-21 cell. Expression of YFP was tested by the fluorescence microscopy and flow cytometer.</p><p><b>RESULTS</b>(1) After the two replicons RNA were transfected into BHK-21 cell, as time went by, the quantity of RNA increased. (2) After RNA of the replicons with YFP were transfected into BHK-21 cell, increasing trend of fluorescent signal and rate of YFP positive cell was observed and tested.</p><p><b>CONCLUSION</b>Full delta prM/E Replicon and Partial delta prM/E Replicon have the ability to duplicate itself and express the foreign protein.</p>


Subject(s)
Animals , Cricetinae , Cell Line , DNA Replication , Encephalitis Virus, Japanese , Genetics , Metabolism , Genetic Engineering , Genetic Vectors , Genetics , Metabolism , Replicon
20.
Chinese Journal of Experimental and Clinical Virology ; (6): 424-426, 2009.
Article in Chinese | WPRIM | ID: wpr-325523

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the interaction of PD-1 and PD-L1 recombination protein and to know their bioactivity and affinity.</p><p><b>METHODS</b>Stick the PD-1 protein on the surface of CM5 sensor chip by the method of Ammine coupling after being preconsentrated. Dilute the PD-L1 protein step by step and reject it to the passage on CM5 sensor chip which had been stick by PD-1. The time of combination is 3 minutes and of separation is 15 minutes, respectively. Observe the procession and analyze data by BIA Evaluation software 4.</p><p><b>RESULTS</b>On the consistency of 40 microg/ml, pH 4.5, the PD-1 protein could couple steady on the surface of CM5sensor chip, RU is 3300. On the density of 200 mmol/ml PD-L1 could combine with PD-1 specifically, RU = 150, K(D) = 3.5 x 10(-6).</p><p><b>CONCLUSION</b>The PD-1 and PD-L1 recombination protein which we expressed by prokaryotic system have good affinity and bioactivity. The results could provide basic condition for later study.</p>


Subject(s)
Humans , Antigens, CD , Chemistry , Genetics , Metabolism , Apoptosis Regulatory Proteins , Chemistry , Genetics , Metabolism , B7-H1 Antigen , Escherichia coli , Genetics , Metabolism , Gene Expression , Kinetics , Programmed Cell Death 1 Receptor , Protein Binding , Recombinant Proteins , Chemistry , Genetics , Metabolism
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