Heterologous expression, purification and characterization of the influenza A virus M2e gene fused to Mycobacterium tuberculosis HSP70(359-610) in prokaryotic system as a fusion protein.
One of the concerns about influenza A vaccine based on M2e protein is their limited potency; hence, optimal approaches to enhance immunogenicity of M2e protein immunization remain to be established. It seems by linking this M2e-peptide to an appropriate carrier such as mycobacterium tuberculosis C-terminal 28-kDa domain of HSP70 (HSP70(359-610)), we can render it very immunogenic. According to previous reports, this study was designed to produce a novel influenza A virus recombinant fusion protein consisted of M2e, a potent immunogenic protein from influenza A virus, fused to C-terminal domain of mycobacterium tuberculosis HSP70, HSP70(359-610), as a carrier and adjuvant. We fused the genes of M2e and HSP70 ( 359-610 ) then inserted in pQE-60, prokaryotic expression vector. This recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M-15. The recombinant fusion protein was purified by Ni-NTA affinity chromatography under denaturing conditions, followed by urea gradient dialysis. The purified fusion protein was analyzed on SDS-PAGE. Western blot assay was used to examine the immunoreaction of the expressed protein using commercial penta-His HRP conjugate antibody. The antigenicity and biological activity of the recombinant protein was also qualitatively detected on the infected MDCK cells surface by immunofluorescence and cell-ELISA assay using rabbit's immunized antiserum. This observation suggest that the expressed fusion protein is useful as a universal recombinant vaccine for overcoming highly mutational influenza virus, but more immunological study in animal lab remains to be evaluated.
Ebrahimi
Seyyed Mahmoud
SM
Department of Biotechnology, Razi Vaccine and Serum Research Institute, P.O. Box 31975/148, Karaj, Tehran, Iran. smebrahimi@shirazu.ac.ir
Tebianian
Majid
M
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005814:Genes, Viral / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D009980:Influenza A virus / Q000235:genetics*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D011387:Prokaryotic Cells / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins