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Marlita S. Pt. Production of Influenza H1N1 Virus Like Particle (VLP) by Baculovirus Expression Vector System. Master's Degree(Biotechnology). King Mongkut's University of Technology Thonburi. KMUTT Library. : King Mongkut's University of Technology Thonburi, 2010.
Production of Influenza H1N1 Virus Like Particle (VLP) by Baculovirus Expression Vector System
Name: Marlita S. Pt
keyword:
Influenza vaccine
Abstract:
Influenza infection is one of the most devastating diseases for human and
animals, which is best prevented through vaccination. Currently, licensed influenza
vaccines are produced in embryonic chicken egg and these have some drawbacks
including contra-indication in people with egg allergies, long duration for vaccine
production, limitation of specific egg supply, biosafety concern in handling infectious
virus, etc. To address the need for safer influenza vaccines and more effective
production process, we have engineered influenza virus like particle (VLP) as an
alternative influenza vaccine. VLP consists of structural proteins of influenza virus with
structure and morphology mimics the virus itself but does not contain any genetic
materials therefore it is non-infectious and production processes are much simpler.
Furthermore, it has been reported that VLP based vaccines have a broader immunogenic
efficiency and better protection than the egg based vaccine. In this study, the influenza
H1N1 VLP was generated by expressing three structural genes of influenza A virus i.e.
hemagglutinin (HA), neuraminidase (NA) and matrix Ml genes using Baculoviruses
Expression Vector System (BEVS). Those three genes were inserted into the
baculovirus genome as confirmed by PCR analysis and then the recombinant
baculovirus was produced and taken to infect into Sf-9 insect cells. Upon infection of
Sf-9 cells, the three genes were co-expressed as observed by RT-PCR analysis. The
presence of recombinant proteins produced by the infected insect cell in the culture
supernatant that were expected to form VLP then analyzed. Western blot analysis using
monoclonal antibody specific to HA protein revealed a band of approximately 70 kDa
which is correspondent to hemagglutinin protein. The recombinant HA protein size is a
little higher than the expected size from the amino acid sequences analysis. This
possibly due to the glycosylation of the recombinant HA protein as post-translational
modification performed by infected insect cells. Furthermore, hemagglutination activity
was also observed. The infected cell culture supernatant was found to be able to
agglutinate the goose red blood cells. In addition, neuraminidase (NA) activity based on
the ability of neuraminidase enzyme in cleaving the sialic acid binding of MUNANA
(4-methylumbelliferyl-N-acetyl-a-D-neuraminic acid) synthetic substrate was 0.12+-0.04
U/ml. These results demonstrate that the BEVS can be used to produce recombinant
proteins of HI NI influenza virus with two main important activities of
hemagglutination and neuraminidase. Whether or not the VLP is formed must be further
investigated by visualization using electron microscope.
King Mongkut's University of Technology Thonburi. KMUTT Library
Address:
BANGKOK
Email:
info.lib@mail.kmutt.ac.th
Name: Kanokwan Poomputsa
Role:
advisor
Created:
2010
Modified:
2011-09-12
Issued:
2011-09-11
วิทยานิพนธ์/Thesis
application/pdf
CallNumber:
BIT634
eng
DegreeName: Master of Science
Level: Master's Degree
Descipline: Biotechnology
RightsAccess:
ใช้เวลา
0.035876 วินาที
Marlita S. Pt
| Title | Contributor | Type |
|---|---|---|
| Production of Influenza H1N1 Virus Like Particle (VLP) by Baculovirus Expression Vector System
มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี Marlita S. Pt | Kanokwan Poomputsa | วิทยานิพนธ์/Thesis |
Kanokwan Poomputsa