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 StandardsABSTRACT
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 ReactionABSTRACT
<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 , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the ability of dengue virus recombinant envelope protein domain expressed in E. coli to inhibit virus infection and induce the neutralizing antibody.</p><p><b>METHODS</b>E III protein of Dengue virus serotypes 1-4 were expressed in E. coli BL21(DE3) then purified. Recombinant proteins were tested to inhibit DV2 from infecting BHK-21 cell. Rabbits were immunized with recombinant proteins to produce anti-E III serum. Antibody titers were determined by neutralizing assay.</p><p><b>RESULTS</b>The recombinant E III proteins of Dengue virus serotypes 1-4 were expressed in E. coli. They effectively protected BHK cells in culture against DV2 infection. All four type anti-E III sera can neutralize DV2 but their efficacies are different.</p><p><b>CONCLUSION</b>proteins of dengue virus expressed in E. coli can directly inhibit DV2 infection. Neutralizing antibodies were induced by E III proteins. Both E III protein of dengue virus and the neutralizing antibodies they induced are more efficient in inhibiting homologous dengue serotypes infection than heterologous serotypes.</p>
Subject(s)
Animals , Cricetinae , Humans , Rabbits , Antibodies, Viral , Allergy and Immunology , Cell Line , Dengue , Allergy and Immunology , Virology , Dengue Virus , Chemistry , Genetics , Allergy and Immunology , Physiology , Down-Regulation , Escherichia coli , Genetics , Metabolism , Immunization , Mesocricetus , Protein Structure, Tertiary , Recombinant Proteins , Chemistry , Genetics , Allergy and Immunology , Viral Envelope Proteins , Chemistry , Genetics , Allergy and Immunology , Virus ReplicationABSTRACT
<p><b>OBJECTIVE</b>To study the effect of Wenmaitong (WMT) and its disassembled formulas on the adhesion of monocytes to endothelial cells induced by hyperlipidemic serum to explore the mechanism of WMT on early arteriosclerosis obliterans (ASO).</p><p><b>METHODS</b>Serums containing whole WMT and its disassembled formulas, including the formula consisted of warming Jing and boosting qi part (Wenjin Yiqi, WY) and that of promoting blood circulation part (Huoxue Tongmai, HT), as well as the serum contained high concentration of lipids were prepared conventionally, respectively. The adhesion of monocytes cell strain THP-1 to human umbilical vascular endothelial cells (HUVEC) was determined by rose bengal stain method, and ELISA was used to detect expressions of intercellular adhesion molecule (ICAM-1), vascular cellular adhesion molecule (VCAM-1) and P-selectin on HUVEC surface.</p><p><b>RESULTS</b>WMT could inhibit THP-1 to HUVEC adhesion induced by hyperlipidemic serum, and down-regulate the expression of ICAM-1, VCAM-1, P-selectin on HUVEC surface, the two disassembled formulas could down-regulate different adhesion molecules.</p><p><b>CONCLUSION</b>One mechanism of WMT on ASO may be its inhibition on arteriosclerosis by way of down-regulating the expression of vascular endothelial cells adhesion molecules to decrease the adhesion of monocyte to VEC, therefore to inhibit the monocytes migrating into vascular intima to develop foam cells.</p>