RESUMO
<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>
Assuntos
Animais , Humanos , Camundongos , Clonagem Molecular , Escherichia coli , Genética , Metabolismo , Expressão Gênica , Imunização , Virus da Influenza A Subtipo H5N1 , Genética , Alergia e Imunologia , Influenza Humana , Alergia e Imunologia , Proteínas Recombinantes , Genética , Alergia e Imunologia , Proteínas da Matriz Viral , Genética , Alergia e ImunologiaRESUMO
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.
Assuntos
Animais , Feminino , Camundongos , Anticorpos Antivirais , Sangue , Células COS , Chlorocebus aethiops , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Genética , Alergia e Imunologia , Imunoglobulina G , Sangue , Virus da Influenza A Subtipo H5N1 , Alergia e Imunologia , Vacinas contra Influenza , Alergia e Imunologia , Camundongos Endogâmicos BALB C , Vacinas de DNA , Alergia e ImunologiaRESUMO
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.
Assuntos
Animais , Humanos , Masculino , Camundongos , Artrite Reumatoide , Terapêutica , Colágeno Tipo II , Células Endoteliais , Metabolismo , Escherichia coli , Genética , Metabolismo , Etanercepte , Terapia Genética , Imunoglobulina G , Genética , Usos Terapêuticos , Camundongos Endogâmicos DBA , Receptores do Fator de Necrose Tumoral , Genética , Usos Terapêuticos , Proteínas Recombinantes de Fusão , Genética , Usos Terapêuticos , Transfecção , Fator de Necrose Tumoral alfa , MetabolismoRESUMO
<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>