Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 24
Filter
1.
West China Journal of Stomatology ; (6): 434-440, 2021.
Article in English | WPRIM | ID: wpr-887755

ABSTRACT

OBJECTIVES@#To investigate the expression of Ki-67, Cyclin D1, P53, and P16 in patients with oral leukoplakia (OLK) and OLK cancerization who have aspicy diet in Chengdu.@*METHODS@#Thirtypatients with OLK andspicy diet and 15 patients with OLK without spicy diet in Chengdu were divided into three groups: hyperplastic OLK (OLK-), OLK with mild to moderate dysplasia (OLK+), and severe dysplastic  OLK or oral squamous cell carcinoma (OSCC) transforming from OLK (OLK++/OSCC). The expression of Ki-67, Cyclin D1, P53, and P16 were detected by immunohistochemistry and statistically analyzed.@*RESULTS@#The expression of Ki-67 and P53 in patients with or without spicy diet in the OLK+and OLK++/OSCC groups were stronger than that of the OLK- group (@*CONCLUSIONS@#Spicy diet did not have an influence on the expression of Ki-67, Cyclin D1, P53, and P16 in patients with OLK and OSCC. The expression of Ki-67, Cyclin D1, and P53 increased with the development of OLK, whereas P16 showed opposite expression trend.


Subject(s)
Humans , Carcinoma, Squamous Cell , Cyclin D1 , Diet , Head and Neck Neoplasms , Ki-67 Antigen , Leukoplakia, Oral , Mouth Neoplasms , Tumor Suppressor Protein p53
2.
West China Journal of Stomatology ; (6): 297-300, 2020.
Article in Chinese | WPRIM | ID: wpr-827542

ABSTRACT

OBJECTIVE@#To explore the treatment conditions of acid decalcified specimens and improve the poor quality of sections and unclear structure of hematoxylin-eosin (HE) staining caused by the change in pH in tooth and hard tissue after acid decalcification.@*METHODS@#A total of 20 cases of oral pathological specimens that contain hard tissues were decalcified and treated with routine treatment, concentrated ammonia water immersion treatment, and saturated lithium carbonate solution immersion treatment. The quality and HE staining effects of hard tissue sections treated with different methods were compared.@*RESULTS@#Compared with routine treatment, lithium carbonate saturated solution treatment showed complete sections. Hematoxylin is strongly stained, the nucleus is clear, and the cytoplasm is bright.@*CONCLUSIONS@#Soaking acid decalcified specimens in lithium carbonate saturated solution before embedding in dehydration can neutralize the acidic environment of the tissue. The quality of sections and HE staining effect are improved and are suitable for the pretreatment of acid decalcified tissue samples of oral pathology.


Subject(s)
Eosine Yellowish-(YS) , Hematoxylin , Staining and Labeling , Tooth
3.
Chinese Medical Journal ; (24): 2183-2189, 2017.
Article in English | WPRIM | ID: wpr-249015

ABSTRACT

<p><b>Background:</b>Portal-vein stent combined with one iodine-125 (I) seed strand has become a new treatment for portal vein tumor thrombosis. However, dosimetric aspects of this irradiation stent have not been reported. Therefore, we aimed to undertake dosimetric analyses comparing portal-vein stents combined with different numbers ofI seed strands.</p><p><b>Methods:</b>A water cylinder was created by a treatment-planning system to simulate a portal-vein stent. The stent was combined with one, two, or threeI seed strands (Groups I, II, and III, respectively). At different prescribed doses (PDs),I seeds of identical activities were loaded on Groups I-III. Conformation number (CN), external volume index, and homogeneity index were calculated. Linear regression analyses were used to evaluate the obtained data.</p><p><b>Results:</b>For identicalI seed activity, when theI seed strand increased from one chain to two, D(dose delivered to 90% of the target volume) increased by ≥184%; when it increased from two chains to three, Dincreased by ≥63%. When the PD was 105 Gy andI seed strands increased from one chain to two, V(percentage of the target volume receiving ≥90% of the PD) increased by 158-249%; when it increased from two chains to three, Vincreased by 7-175%. CN was correlated positively withI seed activity (B = 0.479, P < 0.001) and number ofI seed strands (B = 0.201, P < 0.001) and was independent of PD (B = -0.002, P = 0.078).</p><p><b>Conclusions:</b>A portal-vein stent combined with a singleI seed strand could not meet dosimetry requirements. For a stent combined with twoI seed strands, when the PD was 105 Gy and seed activity was 0.7 mCi, the dose distribution could satisfy dosimetry requirements. For a stent combined with threeI seed strands, if the PD was 105, 125, or 145 Gy, the recommended seed activities were 0.5, 0.5, and 0.6 mCi, respectively.</p>

4.
Chinese Medical Journal ; (24): 1093-1099, 2017.
Article in English | WPRIM | ID: wpr-266856

ABSTRACT

<p><b>BACKGROUND</b>A novel radioactive 125I seed-loaded biliary stent has been used for patients with malignant biliary obstruction. However, the dosimetric characteristics of the stents remain unclear. Therefore, we aimed to describe the dosimetry of the stents of different lengths - with different number as well as activities of 125I seeds.</p><p><b>METHODS</b>The radiation dosimetry of three representative radioactive stent models was evaluated using a treatment planning system (TPS), thermoluminescent dosimeter (TLD) measurements, and Monte Carlo (MC) simulations. In the process of TPS calculation and TLD measurement, two different water-equivalent phantoms were designed to obtain cumulative radial dose distribution. Calibration procedures using TLD in the designed phantom were also conducted. MC simulations were performed using the Monte Carlo N-Particle eXtended version 2.5 general purpose code to calculate the radioactive stent's three-dimensional dose rate distribution in liquid water. Analysis of covariance was used to examine the factors influencing radial dose distribution of the radioactive stent.</p><p><b>RESULTS</b>The maximum reduction in cumulative radial dose was 26% when the seed activity changed from 0.5 mCi to 0.4 mCi for the same length of radioactive stents. The TLD's dose response in the range of 0-10 mGy irradiation by 137Cs γ-ray was linear: y = 182225x - 6651.9 (R2=0.99152; y is the irradiation dose in mGy, x is the TLDs' reading in nC). When TLDs were irradiated by different energy radiation sources to a dose of 1 mGy, reading of TLDs was different. Doses at a distance of 0.1 cm from the three stents' surface simulated by MC were 79, 93, and 97 Gy.</p><p><b>CONCLUSIONS</b>TPS calculation, TLD measurement, and MC simulation were performed and were found to be in good agreement. Although the whole experiment was conducted in water-equivalent phantom, data in our evaluation may provide a theoretical basis for dosimetry for the clinical application.</p>


Subject(s)
Humans , Brachytherapy , Methods , Computer Simulation , Monte Carlo Method , Radiometry , Methods , Thermoluminescent Dosimetry , Methods
5.
Journal of Southern Medical University ; (12): 1301-1307, 2017.
Article in Chinese | WPRIM | ID: wpr-299358

ABSTRACT

<p><b>OBJECTIVE</b>To explore the role of the receptor for advanced glycation end products (RAGE) in regulating the expression of MUC5AC and mucus production in a mouse model of toluene diisocyanate (TDI)?induced asthma.</p><p><b>METHODS</b>BALB/c mice were randomly divided into control group, vehicle (AOO) group, TDI?induced asthma group and RAGE inhibitor (FPS?ZM1) group. PAS staining, Western blotting, and immunohistochemistry were used to analyze the changes in mucus production and MUC5AC expression in the airway of the mice, and the expression of p?ERK was detected with Western blotting. In vitro cultured human bronchial epithelial cell line 16HBE was transfected with lentiviral vector carrying short hairpin RNA targeting RAGE (shRNA?RAGE) and subsequently challenged with a TDI?human serum albumin (TDI-HSA) conjugate, and the changes in cellular MUC5AC mRNA expression as detected using RT-PCR; the protein expressions of ERK and p?ERK in the cells were examined with Western blotting. The effect of ERK inhibitor U0126 pretreatment on MUC5AC mRNA expression was also analyzed in the cells.</p><p><b>RESULTS</b>Compared with the control mice, TDI-induced asthmatic mice showed significantly higher rates of PAS positivity and increased MUC5AC and p?ERK expressions in the airway (P<0.05). Treatment with FPS?ZM1 significantly decreased PAS positivity and lowered MUC5AC and p?ERK expressions in the airway of the asthmatic mice (P<0.05). Exposure of 16HBE cells to TDI?HSA caused a significant increase in MUC5AC mRNA expression and p?ERK protein expression (P<0.05), while RAGE knockdown obviously suppressed TDI?HSA-induced upregulation of p-ERK and MUC5AC mRNA (P<0.05). Treatment with the ERK inhibitor U0126 also lowered TDI?HSA?induced up?regulation of MUC5AC mRNA in the cells (P<0.05).</p><p><b>CONCLUSION</b>RAGE signaling induces MUC5AC expression via extracellular signal-regulated kinase pathway to promote mucus overproduction in mice with TDI-induced asthma.</p>

6.
Journal of Southern Medical University ; (12): 932-935, 2016.
Article in Chinese | WPRIM | ID: wpr-286871

ABSTRACT

<p><b>OBJECTIVE</b>To evaluate fractional exhaled nitric oxide (FENO) level in patients with subacute cough and its value in predicting the patients' response to inhaled corticosteroids (ICS) treatment.</p><p><b>METHODS</b>A total of 100 patients with persistent cough lasting more than 3 weeks were enrolled, including 52 patients with subacute cough and 48 with chronic cough. FENO, spirometry, and responses to ICS therapy of the patients were evaluated.</p><p><b>RESULTS</b>The recruited patients had a median (inter-quartile ranges) FENO level of 19 ppb (12-30 ppb). Patients with chronic cough had a significantly higher median FENO level than those with subacute cough (20.5 vs 16 ppb; Z=-2.245, P=0.025). A FENO level ≥25 ppb was recorded in 15 (28.8%) patients with subacute cough, as compared with 20 (41.6%) in patients with chronic cough (χ(2)=1.801, P=0.179). With a FENO ≥25 ppb as the critical value to justify ICS treatment, 15 patients with subacute cough received ICS and 14 (93.3%) of them showed obvious relief of cough after 2 weeks of therapy, a response rate similar to that of 85.0% (17/20) in patients with chronic cough receiving the treatment (χ(2)=0.588, P=0.443). In patients with subacute cough, those with cough variant asthma (CVA) or eosinophilic bronchitis (EB) had a significantly higher median FENO level than those with postinfectious cough [(16 (11-31) ppb vs 11 (8-19) ppb, P<0.01]. In the etiological analysis, CVA or EB was identified in 23 (44.2%) of the patients with subacute cough, as compared 21 (43.8%) in patients with chronic cough (χ(2)=0.002, P=0.961).</p><p><b>CONCLUSION</b>FENO may be an important indicator for etiological diagnosis of subacute cough and for predicting the response to ICS treatment.</p>


Subject(s)
Female , Humans , Male , Adrenal Cortex Hormones , Therapeutic Uses , Breath Tests , Chronic Disease , Cough , Diagnosis , Drug Therapy , Exhalation , Nitric Oxide
7.
Chinese Journal of Experimental and Clinical Virology ; (6): 81-84, 2013.
Article in Chinese | WPRIM | ID: wpr-318098

ABSTRACT

<p><b>OBJECTIVE</b>To construct the recombinant baculovirus with NA gene of Influenza H1N1 virus.</p><p><b>METHODS</b>Full-length NA gene of Influenza virus H1N1 (A/PR/8/34) was amplified by PCR and inserted into pFastBacdual vector to construct the recombinant baculovirus transfer vector pFBD-NA. Recombinant shuttle vectors rBacmid-NA was then obtained after transforming DH10B competent cells containing bacmid plasmids. After transfecting into sf9 cells, recombinant baculovirus rBac-NA was obtained. The rBac-NA genome was extracted and identified by PCR. NA protein expressed by recombinant baculovirus-infected sf9 cells was determined by IFA, Western Bolt and ELISA.</p><p><b>RESULTS</b>PCR results proved that recombinant shuttle vectors rBacmid-NA was successfully constructed. NA protein was detected by IFA and showed strong specific green fluorescence on the surface of infected cells. NA protein was recognized by two polyclonal antibodies specific for NA in Western Blot. ELISA showed specific reaction of recombinant NA protein with mouse polyclonal antibody against influenza virus (PR8), indicating high antigenicity.</p><p><b>CONCLUSION</b>Recombinant baculovirus rBac-NA that expresse NA protein of influenza virus was successfully constructed. This work provides a basis for further study on NA protein function and novel influenza vaccine development.</p>


Subject(s)
Animals , Mice , Baculoviridae , Genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression , Genetic Vectors , Influenza A Virus, H1N1 Subtype , Genetics , Influenza Vaccines , Neuraminidase , Genetics , Recombinant Proteins , Spodoptera
8.
Chinese Journal of Experimental and Clinical Virology ; (6): 141-143, 2013.
Article in Chinese | WPRIM | ID: wpr-318080

ABSTRACT

<p><b>OBJECTIVE</b>To discuss the enzyme linked immune spot test (ELISPOT) detected the cellular immune response induced by human Bocavirus (HBoV) VP2 virus-like particles (VLPs).</p><p><b>METHODS</b>After immunized by HBoV VP2 VLPs, the specific cellular immune response in mice were detected by ELISPOT assay, observe the ELISPOT results at the conditions of different polypeptide stimulate, different cell culture time, different cell concentration and different specific stimulus peptide concentration, then screening the right ELISPOT experimental conditions and establish the ELISPOT method.</p><p><b>RESULTS</b>The spots induced by HBoV1 VLPs immunized mice spleen lymphocytes stimulate with polypeptide P3 (GYIPIENEL) and P5 (LYQMPFFLL)were 233 spots/10(6) cells and 157 spots/10(6) cells,spots induced by HBoV2 VLPs immunized mice spleen lymphocytes stimulate with polypeptide P8 (GYIPVIHEL) were 113 spots/10(6) cells; 24 hours is the best time for culture, at this time HBoV1 and HBoV2 groups specificity secretion IFN-gamma ratio were 232 spots/10(6) cells and 119/10(6) cells; Best concentration of mice spleen lymphocyte is 5 x 10(5), right now HBoV1 and HBoV2 group specificity secretion IFN-gamma ratio were 232 spots/10(6) cells and 108/10(6) cells; Best concentration of polypeptides is 10 microg/ml, HBoV1 and HBoV2 group specificity secretion IFN -gamma ratio were 233 spots/10(6) cells and 96/10(6) cells.</p><p><b>CONCLUSIONS</b>HBoV1 and HBoV2 specificT-cell epitope in BABL/c mice were P3, P5 (HBoV1) and P8 (HBoV2). The best experiment condition were: cell cultivated for 24 h, cells concentration for 5 x 10(5) cells/well, stimulating polyperides concentration for 10 microg/ml, it can use to study the cellular immune induced by HBoV in mice.</p>


Subject(s)
Animals , Male , Mice , Amino Acid Sequence , Enzyme-Linked Immunospot Assay , Methods , Epitopes, T-Lymphocyte , Human bocavirus , Allergy and Immunology , Immunity, Cellular , Interferon-gamma , Mice, Inbred BALB C , Molecular Sequence Data , Virion , Allergy and Immunology
9.
Chinese Journal of Virology ; (6): 7-11, 2013.
Article in Chinese | WPRIM | ID: wpr-339982

ABSTRACT

To establish a mammalian cell line for stable expression of the matrix protein 2 (M2) of influenza virus type A. M2 gene was amplified by PCR from the influenza virus strain A/PR/8/34. The PCR product was cloned into eukaryotic expression vector pcDNA5/FRT. After identification with restriction enzyme digestion, the plasmid was co-transfected with plasmid pOG44 which expressed Flp in Flp-In-CHO cells. The target gene was integrated into chromosome of CHO cells by homologous recombination in vivo. Recombinant CHO-M2 cell lines were selected for hygromycin B resistance. A total of 15 recombinant cell strains with high expression of M2 protein were screened by hygromycin, and the expression of M2 protein was determined by IFA and Western blot. After subculturing for 10 passages, the presence of M2 gene in the CHO-M2 cells was confirmed by PCR, and the expression of M2 protein were proved by IFA and Western blot. We successfully constructed a mammalian cell line which stably expressed M2 protein of influenza virus type A. The cell line will be useful for studies on function of M2 protein and provide tools for novel influenza virus vaccine development.


Subject(s)
Animals , Cricetinae , CHO Cells , Cell Culture Techniques , Cell Line , Cricetulus , Influenza A Virus, H2N2 Subtype , Chemistry , Recombinant Proteins , Viral Matrix Proteins , Genetics
10.
Chinese Journal of Virology ; (6): 231-236, 2012.
Article in Chinese | WPRIM | ID: wpr-354742

ABSTRACT

The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines.


Subject(s)
Animals , Baculoviridae , Genetics , Metabolism , Cell Line , Cloning, Molecular , Gene Expression , Genetic Vectors , Genetics , Metabolism , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Allergy and Immunology , Influenza A Virus, H1N1 Subtype , Genetics , Allergy and Immunology , Spodoptera , Transfection , Viral Matrix Proteins , Genetics , Allergy and Immunology
11.
Chinese Journal of Experimental and Clinical Virology ; (6): 167-169, 2011.
Article in Chinese | WPRIM | ID: wpr-231161

ABSTRACT

<p><b>OBJECTIVE</b>To construct vectors expressing M2 and NA genes of H5N1 influenza virus.</p><p><b>METHODS</b>Based on the human H5N1 avian influenza virus (A/Anhui/1/2005) isolated in china, M2 and NA genes were amplified by PCR. M2 or NA gene was subcloned into pStar vector to construct recombinant pStar-M2/, pStar-/M2, pStar-NA/and pStar-NA/. Furthermore, both of the M2 and NA genes were subcloned into pStar to construct two genes co-expressing recombinant pStar-M2/NA and pStar-NA/M2. Expression of the genes were detected by IFA after transfection of 293 cells with the recombinant plasmids.</p><p><b>RESULTS</b>Recombinant plasmids were constructed and identified by restriction endonuclease digestion. Expression of the genes cloned into the recombinant plasmids was confirmed by IFA.</p><p><b>CONCLUSION</b>Recombinant plasmids expressing M2 and/or NA genes of H5N1 influenza virus were constructed, which provided basis for development of influenza DNA vaccine.</p>


Subject(s)
Humans , Cell Line , Genetic Vectors , Genetics , Influenza A Virus, H5N1 Subtype , Genetics , Metabolism , Neuraminidase , Genetics , Metabolism , Plasmids , Genetics , Viral Matrix Proteins , Genetics , Metabolism , Viral Proteins , Genetics , Metabolism
12.
Chinese Journal of Experimental and Clinical Virology ; (6): 254-257, 2011.
Article in Chinese | WPRIM | ID: wpr-246253

ABSTRACT

<p><b>OBJECTIVE</b>To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein.</p><p><b>METHODS</b>M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1.</p><p><b>RESULTS</b>The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals.</p><p><b>CONCLUSION</b>These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.</p>


Subject(s)
Animals , Humans , Mice , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Gene Expression , Immunization , Influenza A Virus, H5N1 Subtype , Genetics , Allergy and Immunology , Influenza, Human , Allergy and Immunology , Recombinant Proteins , Genetics , Allergy and Immunology , Viral Matrix Proteins , Genetics , Allergy and Immunology
13.
Chinese Journal of Virology ; (6): 189-194, 2010.
Article in Chinese | WPRIM | ID: wpr-297885

ABSTRACT

M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.


Subject(s)
Animals , Female , Mice , ADP Ribose Transferases , Genetics , Bacterial Toxins , Genetics , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Genetics , Exotoxins , Genetics , Gene Expression , Immunization , Influenza A virus , Allergy and Immunology , Physiology , Lung , Allergy and Immunology , Virology , Mice, Inbred BALB C , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Viral Matrix Proteins , Genetics , Allergy and Immunology , Virulence Factors , Genetics
14.
Chinese Journal of Experimental and Clinical Virology ; (6): 412-414, 2009.
Article in Chinese | WPRIM | ID: wpr-325527

ABSTRACT

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies specific for M1 protein of H5N1 subtype human influenza virus, this work may provide new tool in rapid diagnosis and study of type A influenza virus.</p><p><b>METHODS</b>BALB/c mice were immunized with purified recombinant H5N1 (A/Anhui/1/2005)/M1 protein expressed in E. coli. Spleen cells of the immunized mice were fused with sp2/0 cells to produce hybridoma cell lines. ELISA was performed to identify the monoclonal antibody against M1 protein of H5N1. Immunofluorescence assay (IFA) were applied to identify the specificity of these antibodies.</p><p><b>RESULTS</b>Three hybridoma cell lines steadily secreting anti-H5N1/M1 McAb were obtained, and their cross reactivity was confirmed by cross-reaction test and IFA.</p><p><b>CONCLUSION</b>Monoclonal antibodies immunized with H5N1 subtype influenza virus M1 protein are cross-reactive, which can be used to detect different subtype of influenze virus type A.</p>


Subject(s)
Animals , Dogs , Humans , Mice , Antibodies, Monoclonal , Allergy and Immunology , Antibodies, Viral , Allergy and Immunology , Cell Line , Cross Reactions , Influenza A Virus, H5N1 Subtype , Genetics , Allergy and Immunology , Influenza, Human , Allergy and Immunology , Virology , Mice, Inbred BALB C , Viral Matrix Proteins , Genetics , Allergy and Immunology
15.
Chinese Journal of Virology ; (6): 107-112, 2009.
Article in Chinese | WPRIM | ID: wpr-334738

ABSTRACT

Based on the human H5N1 influenza virus strain A/Anhui/1/2005, recombinant adenovirus co-expressing M1 and HA genes of H5N1 influenza virus was constructed using an internal ribosome entry site (IRES) sequence to link the two genes. The M1 and HA genes of H5N1 influenza virus were amplified by PCR and subcloned into pStar vector separately. Then the M1-IRES-HA fragment was amplified and subcloned into pShuttle-CMV vector, the shuttle plasmid was then linearized and transformed into BJ5183 bacteria which contained backbone vector pAd-Easy. The recombinant vector pAd-Easy was packaged in 293 cells to get recombinant adenovirus Ad-M1/HA. CPE was observed after 293 cells were transfected by Ad-M1/HA. The co-expression of M1 and HA genes was confirmed by Western-blot and IFA (immunofluorescence assay). The IRES containing recombinant adenovirus allowed functional co-expression of M1 and HA genes and provided the foundation for developing new influenza vaccines with adenoviral vector.


Subject(s)
Humans , Adenoviridae , Genetics , Metabolism , Antibodies, Viral , Gene Expression , Genetics , Genetic Vectors , Pharmacology , Influenza A Virus, H5N1 Subtype , Influenza Vaccines , Plasmids , Pharmacology , Recombinant Fusion Proteins , Genetics , Metabolism , Viral Vaccines , Pharmacology
16.
Chinese Journal of Virology ; (6): 366-370, 2007.
Article in Chinese | WPRIM | ID: wpr-334881

ABSTRACT

Based on the first isolated human H5N1 influenza virus strain A/Anhui/1/2005 in China, HA and HA1 genes were amplified and cloned into the eukaryotic expression vector pStar. The recombinant plasmids pStar-HA and pStar-HA1 were transfected into COS7 cells. Western blot and IFA showed that the two recombinant DNA plasmids were successfully expressed in eukaryotic cells. BALB/c mice were immunized with the plasmids DNA by intramuscular injection. Anti-HA specific antibody in peripheral blood of immunized mice was tested by ELISA. The results showed that the recombinant plasmids successfully induced the anti-HA humoral immune response, and there was no significant difference between HA and HA1 as immunogen. This work provides basis for future development of novel avian flu vaccine.


Subject(s)
Animals , Female , Mice , Antibodies, Viral , Blood , COS Cells , Chlorocebus aethiops , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Allergy and Immunology , Immunoglobulin G , Blood , Influenza A Virus, H5N1 Subtype , Allergy and Immunology , Influenza Vaccines , Allergy and Immunology , Mice, Inbred BALB C , Vaccines, DNA , Allergy and Immunology
17.
Chinese Journal of Experimental and Clinical Virology ; (6): 8-10, 2006.
Article in Chinese | WPRIM | ID: wpr-305539

ABSTRACT

<p><b>BACKGROUND</b>To analyze the genetic and antigenic characteristics of human H5N1 virus isolated from the mainland of China.</p><p><b>METHODS</b>The hemagglutinin (HA) gene of human H5N1 virus were sequenced and analyzed.</p><p><b>RESULTS</b>The results of HA gene sequencing showed that all the virus isolates belong to the same group because of the high similarity, but they were different from the virus isolated from Thailand and Vietnam. The sequence data also showed that the receptor specificity and the connecting peptide between HA1 and HA2 are still avian influenza origin.</p><p><b>CONCLUSION</b>The virus isolates from mainland of China until now belong to the same group and are different from the virus isolated from Thailand and Vietnam, and there is no evidence showing the human-avian influenza reassortant and recombination.</p>


Subject(s)
Animals , Chick Embryo , Humans , China , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Influenza A Virus, H5N1 Subtype , Classification , Genetics , Influenza, Human , Virology , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA
18.
Chinese Journal of Experimental and Clinical Virology ; (6): 24-26, 2006.
Article in Chinese | WPRIM | ID: wpr-305534

ABSTRACT

<p><b>BACKGROUND</b>To establish a method for H5N1 RNA detection and laboratory diagnosis of suspected human avian influenza (H5N1) virus infected cases.</p><p><b>METHODS</b>The typing and sub-typing primers were designed according to M and H5 and N1 gene respectively, and the RT-PCR and real-time PCR were developed using these primers.</p><p><b>RESULTS</b>The RT-PCR and real-time PCR could be used for H5N1 detection specifically, and there was no cross reaction with other influenza subtypes such as H1 and H3. The sensitivity for RT-PCR and real-time PCR was 1 TCID50 and 0.01 TCID50 respectively. Thirteen laboratory confirmed human H5N1 cases were detected from 42 suspected cases by using these methods.</p><p><b>CONCLUSION</b>The established RT-PCR and real-time PCR methods can be used for laboratory detection of suspected human H5N1 cases.</p>


Subject(s)
Animals , Chick Embryo , Humans , DNA Primers , Genetics , Influenza A Virus, H5N1 Subtype , Genetics , Influenza, Human , Diagnosis , Virology , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Methods , Sensitivity and Specificity , Viral Proteins , Genetics
19.
Chinese Journal of Biotechnology ; (12): 378-383, 2006.
Article in Chinese | WPRIM | ID: wpr-286280

ABSTRACT

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine, acting as a regulator of inflammation and immunity. TNFalpha plays a critical role in the pathogenesis of rheumatoid arthritis. Blocking of TNFa activity suppressed inflammatory tissue damage. In present study, the chimeric gene of soluble TNF receptor and IgG Fc fragment (sTNFR-IgG FC) was cloned into the mammalian cell expression vector pStar. When the plamid pStar/sTNFR-IgGFc-GFP was transfected into endothelial cells, a considerable expression of the sTNFR-IgG Fc fusion protein was detected. Moreover, the product in 100microL expression supernatant could completely antagonize the cytolytic effect of 1ng TNFa on L929 cells, even at 1/64 dilution. Then the plasmid was delivered into CIA-induced rheumatoid arthritis mice by tail vein injection. The expression of sTNFR-IgG Fc was detected in liver by RT-PCR. Animals in treatment group showed reduced symptoms of arthritis and more active. This treatment induced decrease of synovial incrassation and prevented the cartilage destruction of the mice RA model. These results show that tail vein injection is an effective way for gene therapy and sTNFR-IgGFc expression plasmid is potential for the treatment of rheumatoid arthritis.


Subject(s)
Animals , Humans , Male , Mice , Arthritis, Rheumatoid , Therapeutics , Collagen Type II , Endothelial Cells , Metabolism , Escherichia coli , Genetics , Metabolism , Etanercept , Genetic Therapy , Immunoglobulin G , Genetics , Therapeutic Uses , Mice, Inbred DBA , Receptors, Tumor Necrosis Factor , Genetics , Therapeutic Uses , Recombinant Fusion Proteins , Genetics , Therapeutic Uses , Transfection , Tumor Necrosis Factor-alpha , Metabolism
20.
Chinese Journal of Experimental and Clinical Virology ; (6): 182-184, 2005.
Article in Chinese | WPRIM | ID: wpr-333067

ABSTRACT

<p><b>OBJECTIVE</b>To explore the role of BM2 protein in the life cycle of influenza B virus.</p><p><b>METHODS</b>The authors screened human kidney MATCHMAKER cDNA library for new binding partners of BM2 of influenza B virus by using the yeast two hybrid system with truncated BM2 (26-109 aa) as the bait.</p><p><b>RESULTS</b>Six positive plasmids encoding N-acetylneuraminate pyruvate lyase, angiopoietin 3, zinc finger protein 251, ribosomal protein S20, protein arginine N-methyltransferase 1 variant 1 (PRMT) and transcription factor-like 1 (TCFL1) were obtained.</p><p><b>CONCLUSION</b>The results suggest that BM2 may play an important role in the life cycle of influenza B virus.</p>


Subject(s)
Humans , Angiopoietin-like Proteins , Angiopoietins , Genetics , Metabolism , DNA-Binding Proteins , Genetics , Metabolism , Gene Library , Influenza B virus , Genetics , Metabolism , Kidney , Metabolism , Oxo-Acid-Lyases , Genetics , Metabolism , Plasmids , Genetics , Protein Binding , Protein-Arginine N-Methyltransferases , Genetics , Metabolism , Repressor Proteins , Genetics , Metabolism , Ribosomal Proteins , Genetics , Metabolism , Transcription Factors , Genetics , Metabolism , Two-Hybrid System Techniques , Viral Proteins , Genetics , Metabolism , Zinc Fingers , Genetics
SELECTION OF CITATIONS
SEARCH DETAIL