title: Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli.
abstract: Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.
authors.affiliation: Advanced Molecular Biology Laboratory, School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: T
authors.initials: T
authors.lastname: Chee Wei
authors.affiliation: Advanced Molecular Biology Laboratory, School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: A G
authors.initials: AG
authors.lastname: Nurul Wahida
authors.affiliation: Advanced Molecular Biology Laboratory, School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: S
authors.initials: S
authors.lastname: Shaharum
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000302:isolation & purification; D008296:Malaysia; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion
chemical_list: D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins; C109963:hemagglutinin, avian influenza A virus