Optimization of H5N1 Virus-Like Particle Production Using Baculovirus Expression Vector System

Ahmad, Mujahidin (2011) Optimization of H5N1 Virus-Like Particle Production Using Baculovirus Expression Vector System. Magister thesis, Universitas Brawijaya.


English Abstract

Outbreak of H5N1, namely, Birdflu, has caused considerable losses to poultry industry worldwide, and at same time, transpecies infection was imminent, particularly from bird to swine n to man, respectively. WHO recommended vaccine as an effective preventive measure against Birdflu. Conventional influenza vaccine production is demanding since manufacturing involves infectious virus, employs embryonated chicken egg as bioreactor, and requires lengthy time period for production which poses limitation once pandemic is encountered. Several alternative approaches for influenza vaccines have been reported, ranging from protein vaccine expressed in bacteria, yeast, plant and mammalian cells. Baculovirus Expression Vector System (BEVS)-insect cells expression system offers several advantages compared with o r systems in terms of handling, robustness, yield, safety and cost efficiency. Virus-like particles (VLPs) was reported as highly effective types of subunit vaccine that mimic overall structure of virus particles except infectious genetic material. Baculovirus expression vector system (BEVS) is versatile and commonly employed for Virus-like Particle production. This study aimed at characterizaing Sf-9 cell growth and optimizing infection parameters affecting VLP production, i.e., Cell Density at Infection (CDI), Multiplicity of infection (MOI), and Time of Harvest (TOH). Subsequently, effect of anti-stress agents supplementation on VLP production was also investigated using response surface methodology. VLP production was quantitated using dot blot assay and hemagglutination assay. For dot blot assay, VLP level was determined by dot intensity resulted from specific binding of VLP to anti-HA antibody on nitrocellulose membrane. For Hamagglutination Assay (HA assay), VLP activity was determined by agglutination of red blood cell (RBC) by hemagglutinin protein on VLP. Results showed that Sf-9 cells could be cultivated to maximum cell density of 2.6×10 6 cells/ml, with a specific growth rate (μ) and population doubling time (PDT) of 0.027 h -1 and 25.65 hours, respectively. Fur r, VLP production could be attained when infection was accomplished with Cell Density at Infection (CDI) of 1.5×10 6 cells/ml, Multiplicity of infection (MOI) of 5 PFU/cell, and Time of Harvest (TOH) of 5 dpi. It also was found that highest recombinant baculovirus production to be used as an inoculum for future production, could be accomplished under same infection condition, however, at 4 days post infection leading to 6.14×10 7 PFU/ml was obtained. Supplementation of growth medium with R-500 sericin fraction at concentration of 500 ng/ml could promote better Sf-9 cell propagation leading to 17.2% increase in cell density, approximately 3.14×10 6 cells/ml in comparison with control (2.6×10 6 cells/ml). Results on optimization of anti-stress agents using central composite design showed that, for main effect, both α-ketoglutarate and selenium were considered statistically significant (p 0.05), whereas sericin was found to be insignificant (p0.05). Additionally, α-ketoglutarate influenced VLP production negatively, meanwhile, selenium affected VLP production positively. Fur r, α-ketoglutarate×α-ketoglutarate and sericin×sericin were also considered significant (p 0.05) and simultaneously posed negative effect on VLP production. Interactions between α-ketoglutarate and sericin as well as selenium positively affected VLP production (p 0.05). It was fur r found that supplementation of α-ketoglutarate, selenium and sericin at concentration of 11.96 mM, 0.382 and 442.2 ng/ml, yielded maximum VLP production. validation study in shake flask indicated that supplementation of anti-stress agents at optimum concentrations obtained using central composite design led to two folds higher in HA titer in comparison with control (no anti-stress agent addition). Batch cultivation using optimized condition resulted in highest HA titer of approximately 1280 HAU/ml when harvested at 5 and 6 days post infection which was in keeping with result obtained with shake flask culture.

Item Type: Thesis (Magister)
Identification Number: TES/660.63/AHM/o/041102863
Subjects: 600 Technology (Applied sciences) > 660 Chemical engineering and related technologies > 660.6 Biotechnology
Divisions: S2/S3 > Magister Teknologi Hasil Pertanian, Fakultas Teknologi Pertanian
Depositing User: Endro Setyobudi
Date Deposited: 27 Sep 2011 18:32
Last Modified: 27 Sep 2011 18:32
URI: http://repository.ub.ac.id/id/eprint/160097
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