Cloning and Expression of Domain III, prM and NS1 Proteins of Zika Virus for Antigenicity Study

Sylvia Empiang, Andrew (2021) Cloning and Expression of Domain III, prM and NS1 Proteins of Zika Virus for Antigenicity Study. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Zika virus (ZIKV) is a mosquito-borne virus that causes Zika fever and neurological complications such as Guillain-Barré syndrome and congenital microcephaly. ZIKV (family Flaviviridae, genus Flavivirus) is transmitted to humans through the bite of infected mosquitoes. Similar to other flaviviruses, ZIKV is a positive-sense, single-stranded RNA virus and has a 10,794 nucleotide genome. The genome consists of structural and non-structural proteins. The envelope (E) glycoprotein is the major structural protein of flavivirus which plays an important role in virus entry mechanism and is a major target for neutralizing antibodies. The E protein consists of three structurally distinct domains; domain I, domain II and domain III. Domain III (EDIII) of the E protein has shown strong neutralizing properties that can elicit antibodies. Precursor membrane (prM) glycoprotein is one of the structural proteins and it has been shown to form an unusual complex with the EDIII protein. This complex is responsible for virus assembly, fusion and also immunity-inducing of the virus. One of the non-structural proteins of flaviviruses, non-structural 1 (NS1) protein is currently a target for viral biomarkers as it is able to induce antibodies production during Zika infection. In this study, the EDIII, prM and NS1 regions of ZIKV were cloned and expressed using the pET SUMO expression system. Methods such as SDS-PAGE and Western blot were used to analyse the expression of each recombinant proteins. The recombinant fusion proteins were successfully expressed at their approximated molecular weights; EDIII (38.2 kDa), prM (20 kDa), NS1 (dimer 40 kDa and monomer 20 kDa). These proteins were then purified using nickel-affinity column chromatography and the reactivity of purified recombinant proteins were evaluated in immunoblot assay and indirect IgG ELISA. Twenty human serum samples were used in both of the assays and the results of this study showed that the recombinant EDIII, prM and NS1 fusion proteins have different consistencies in the assays.

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