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Expression of a Recombinant Zika Virus E Domain III Protein by E. coli System

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Title:Expression of a Recombinant Zika Virus E Domain III Protein by E. coli System
Authors:Lee, Awapuhi
Contributors:Nerurkar, Vivek (instructor)
Keywords:Zika virus
Date Issued:13 Aug 2019
Abstract:Background: Zika virus (ZIKV) is a positive-sense single-stranded RNA virus that belongs to the genus Flavivirus. ZIKV and dengue virus (DENV) have similar genomes encoding for proteins that make up the virus. The immune system produces antibodies against these proteins and can be cross-reactive between ZIKV and DENV. Protein identity for envelope (E) domain III is different between ZIKV and DENV by protein alignment. Antibodies against ZIKV E domain III are specific to ZIKV and have high neutralizing levels. ZIKV E domain III protein may be useful for differential diagnosis between ZIKV and DENV and could be used for development of a rapid test kit.
Objective: To express a recombinant ZIKV E domain III protein using an E. coli system.
Materials & Methods: A recombinant plasmid was constructed containing ZIKV E domain III gene attached to a six-histidine motif and an antibiotic resistance gene. BL21 (DE3) E. coli was transformed with this recombinant plasmid and plated on a plate with antibiotic. A colony from the plate was picked and cultured in LB broth. After bacteria culture, the recombinant plasmid was extracted and isolated from the bacteria. The isolated plasmid was amplified by polymerase chain reaction (PCR) and analyzed by gel electrophoresis and ethidium bromide staining. The bacteria culture was also used to express the recombinant ZIKV E domain III protein using isopropyl β-D-1-thiogalactopyranoside (IPTG). Protein expression was analyzed by Coomassie blue stain and western blot. Anti-histidine was used to detect the recombinant ZIKV E domain III protein.
Results: Ethidium bromide staining revealed that the recombinant plasmid was present in the bacterial culture. Staining by Coomassie blue showed that the samples contained protein, but the recombinant protein was not detected by western blot when reacted with anti-histidine.
Conclusions: The recombinant ZIKV E domain III protein was not expressed despite using E. coli transformed with the plasmid. Based on the attempted expression of the recombinant ZIKV E domain III protein, developing ZIKV serodiagnostic assays can be challenging.
Rights:Attribution-NonCommercial-NoDerivs 3.0 United States
Appears in Collections: MHIRT Poster Session 2019

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