Seng, Teh Yew
(2013)
Construction and cloning of synthetic
M2e-NP gene from asian influenza a viruses.
Masters thesis, Universiti Sains Malaysia.
Abstract
The sudden emergence and explosive spreading of novel influenza A viruses in the
community represents a major threat to global public health. Conventional influenza
vaccines are mainly targeting two surface glycoproteins namely hemagglutinin (HA)
and neuraminidase (NA) that undergo antigenic drift and antigenic shift
continuously. This poses extraordinary challenges to the virologist for strain
identification and effective vaccine preparation during a pandemic. Hence, there is a
pressing need to develop an effective universal vaccine that can confer immediate
protection against different variants and subtypes of influenza A viruses. In recent
years, nucleoprotein (NP) and extracellular domain of ion channel matrix protein
(M2e) have been widely targeted as possible universal vaccine candidates since these
two proteins are highly conserved among all influenza A viruses. In this study, we
aimed to construct, amplify and clone synthetic M2e-NP gene that can serve as
universal influenza vaccine in future. This study comprises of two major parts. First
part involves in silico analysis of M2e and NP amino acid sequence from 24
subtypes and variants of Asian influenza A viruses in order to identify conserved and
immunogenic peptide sequences. In second part, II newly synthesized
oligonucleotides with length ranged from 32-38 bp were assembled and amplified
using Taq DNA polymerase in assembly PCR. Subsequently, PCR products were
ligated into the pCR®2.1-TOPO® vector followed by transformation into the E. coli
DH5a competent cells. Few white colonies were selected and screened via colony
PCR. Then, recombinant plasmids that harboured desired insert were extracted from
positive clone and subjected to restriction enzyme digestion using Hindlll and Xhol
to verify the presence of the insert prior to sequencing. In the present study, we had
successfully constructed and cloned synthetic M2e-NP gene. Target band with size
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