RESUMO
<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>
Assuntos
Animais , Camundongos , Baculoviridae , Genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vetores Genéticos , Vírus da Influenza A Subtipo H1N1 , Genética , Vacinas contra Influenza , Neuraminidase , Genética , Proteínas Recombinantes , SpodopteraRESUMO
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.
Assuntos
Animais , Baculoviridae , Genética , Metabolismo , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Vetores Genéticos , Genética , Metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Genética , Alergia e Imunologia , Vírus da Influenza A Subtipo H1N1 , Genética , Alergia e Imunologia , Spodoptera , Transfecção , Proteínas da Matriz Viral , Genética , Alergia e ImunologiaRESUMO
<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
<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>