ABSTRACT
<p><b>OBJECTIVE</b>To Construction of P and NP genes eukaryotic expression vectors of Newcastle Disease Virus LaSota strain,study its reverse genetics and functional genome of NDV.</p><p><b>METHODS</b>P, NP genes were amplified and cloned into pGEM-T easy vector and then subcloned into pcDNA3.1 (+) expression vector respectively, the recombinant plasmids were named pcDNA3.1 (+)-P and pcDNA3.1 (+)-NP, Recombinant plasmids were transfected into 293 and BHK-21 cells respectively and were detected using IE and Western blot analysis.</p><p><b>RESULTS</b>Expression of P, NP genes were detected and confirmed by the IE and WB analysis.</p><p><b>CONCLUSION</b>The recombinant eukaryotic plasmids pcDNA3. 1(+)-P, pcDNA3.1 (+)-NP were expressed in 293 and BHK-21 cells successfully. This research may be helpful for further study of reverse genetics and functional genome of NDV.</p>
Subject(s)
Animals , Cricetinae , Humans , Cell Line , Gene Expression , Genetic Vectors , Genetics , Metabolism , Newcastle disease virus , Genetics , Metabolism , Nucleoproteins , Genetics , Metabolism , Phosphoproteins , Genetics , Metabolism , Viral Proteins , Genetics , MetabolismABSTRACT
<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 ImmunologyABSTRACT
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 , PharmacologyABSTRACT
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 ImmunologyABSTRACT
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 , MetabolismABSTRACT
<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 , GeneticsABSTRACT
Tumor rapid growth depends on neovascularization. Vascular endothelial growth factor, the main mediator during the occurrence and formation of vascularization, has specific receptors whose expression rate shows difference of orders of magnitude between tumor and the normal tissue, so it can be used to transport toxin molecules to the proliferative tumor endothelial and kill cancer cells. In our experiment, we constructed fusion protein DT-VEGF by linking diphtheria toxin's forward 389 amino acids's gene and VEGF165 via a linker. DT-VEGF is expressed in E. coli and purified. Our experiment proves in can kill vascular endothelial cells specifically, and the inhibition of neovascularization of chicken chorionic membrane is also confirmed.