Cloning and overexpression of influenza a H1N1 NS1 protein in escherichia coli
Influenza virus is globally pathogenic and it is usually associated with zoonotic respiratory disease. It possesses a lipid-bounded segmented genome which encodes at least one biochemically-distinct protein. Its subtype A can be classified according to antigenic differences. NS1 protein is defined a...
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| Format: | Thesis |
| Language: | English |
| Published: |
2010
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| Subjects: | |
| Online Access: | http://eprints.utm.my/id/eprint/12311/4/OngLihLihMFBB2010.pdf http://eprints.utm.my/id/eprint/12311/ |
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| Summary: | Influenza virus is globally pathogenic and it is usually associated with zoonotic respiratory disease. It possesses a lipid-bounded segmented genome which encodes at least one biochemically-distinct protein. Its subtype A can be classified according to antigenic differences. NS1 protein is defined as nonstructural protein in the virus. It is a known multifunctional virulence factor. It can only be detected in the infected cell. In this study, the NS1A gene was successfully cloned into the BamHI/SacI cleaved-pET- 32c(+) vector and subsequently electro-transformed into E. coli BL21(DE3) expressing host. There were three positive clones confirmed to contain the gene of interest by sequencing. Protein expression in soluble and insoluble fractions was observed in E. coli BL21(DE3). The clone 104 was selected for subsequent analysis. Better NS1A protein expression was found at 37°C by 5mM lactose induction. Purification of the NS1A recombinant protein from the inclusion bodies fraction was attempted by Ni-NTA affinity chromatography and ion exchange chromatography. The physical condition along the purification column and the biological properties of the protein itself may perhaps result in the loss of protein and its corresponding immunogenicity. Ammonium sulfate at 20% saturation was attempted to sufficiently concentrate and partially purify the NS1A recombinant protein. The ammonium sulfate precipitated NS1A recombinant protein has shown significant immno-response to the polyclonal antibody in Western blot. The 37kDa NS1 protein was detected to react with the H1N1 NS polyclonal antibody. |
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