UPSI Digital Repository (UDRep)
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UPSI Digital Repository (UDRep)
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| Abstract : Perpustakaan Tuanku Bainun |
| Newcastle disease virus (NOV) is the only member of the genus Avulavirus of the family
Paramyxoviridae. NOV causes a respiratory disease in birds known as Newcastle disease (ND) which
may result in high mortality in susceptible hosts such as chickens leading to substantial loss in
the poultry industry. Recent outbreak has been reported in many countries including Malaysia. The
continuing treat of ND to the poultry industry requires routine testing through development of
better diagnostic tools. Therefore, the objective of the current study was to express the
immunogenic nucleocapsid (NP) gene in a Pichia pastoris expression system with a view to develop a
potential and cost effective antigen for development of a diagnostic test.
In the present study, the gene encoding NP protein of Newcastle disease virus strain AF2240 was
cloned into expression vector, pPICZA and placed under the control of methanol inducible alcohol
oxidase (AOX) promoter. Then recombinant multi-copy number Pichia cells with Mut phenotype were
selected for NP protein expression.
The optimization of the NP protein production in 50 ml culture was carried out for
methanol concentration and different loaded volume in identical shake flask. A time course study
for NP production in 250-ml flask with the optimized conditions was perfonned as well. The result
showed that NP protein could be detected after 12 h of methanol induction and the level of protein
expression decreased over time. The recombinant NP was purified from the yeast culture using
sucrose gradient ultracentrifugation. The high level and intact recombinant nucleocapsid protein
expression (570 mg/I) was obtained after 24 h of induction with I% methanol when I 0% of the shake
flask was loaded with MMH (minimal methanol with histidine) medium. Western blot analysis using
polyclonal NP antibody confirmed the expression of NP with the molecular weight of 53 kDa
indicating that NP protein retained its antigenicity. The recombinant NP protein was highly stable
in P. pastoris system because there was no degraded product after purification. This result proved
that the yeast expression system produces a high yield of recombinant NP protein. The production of
recombinant NP protein in bulk as the antigen for diagnostic tools would facilitate the monitoring
of NDV infection as well as allowing a more effective control of the disease.
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