Synthesis of double stranded RNA using in vivo and in vitro approaches towards the development of recombinant vaccines in grouper

Chin Lai Mun, Lai Mun (2014) Synthesis of double stranded RNA using in vivo and in vitro approaches towards the development of recombinant vaccines in grouper. Masters thesis, Universiti Malaysia Sabah.

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Abstract

Groupers are a commercially important fisheries resource and have been adopted as a target species by the aquaculture industry. The management of diseases in closed circuit systems remains as a major concern. Grouper Nervous Necrosis Virus (GNNV) is one of the examples of major causative agents of morbidity and mortality that led to heavy economic loss. Therefore, countermeasures such as development of vaccines are important to prevent further damage. RNA vaccines have several major advantages over DNA vaccines and double stranded RNA has been documented to be the key activator of the innate immune response making it a perfect candidate for vaccine development. Hence, this study was directed towards the synthesis of dsRNA using two methods; in vivo and in vitro transcription. The gene construct for GNNV coat protein-pLITMUS was developed for the expression of RNA encoding the coat protein of GNNV when IPTG was induced. Coat protein of GNNV and vector, pLITMUS 28i were isolated manually from TOP 10 stock and ligated using T4 DNA ligase before being cloned using TOP 10 as the bacterial host forming the gene construct, GNNV coat protein-pLITMUS. Subsequently, the expression recombinant DNA; GNNV coat proteinpLITMUS was transformed into E. coli C43 (DE3) and HT115 (DE3) in order to utilize the T7 RNA polymerase expression system present in these strains to produce dsRNA. Production of dsRNA via in vivo bacterial induction system was initiated in E. coli (C43 and HT115) by addition IPTG. Concurrently, dsRNA was also synthesized through in vitro transcription system using T7 Quick High Yield RNA Synthesis Kit. The production of dsRNAs by both approaches was verified via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results in RT-qPCR showed that dsRNA could be synthesized via both methods; in vivo and in vitro where in vitro transcription has showed a higher production rate in both C43 and HT115 with average CT value of 20.28 and 17.94; respectively. On the other hand, in vivo transcription using C43 and HT115 had 22.59 and 22.12; respectively. In conclusion, in vivo transcription appeared to be a better production approach compared to in vitro. Even though in vivo transcription showed slightty lower production rate in RT-qPCR results, the production cost is of in vitro is very much higher compared to in vivo and it is not practical to utilize a costly approach in mass production of dsRNA for a small difference amount in yield.

Item Type:	Thesis (Masters)
Keyword:	Grouper Nervous Necrosis Virus, dsRNA synthesis, RNA vaccine, Aquaculture biotechnology, Viral disease in fish
Subjects:	Q Science > QP Physiology > QP1-(981) Physiology > QP501-801 Animal biochemistry
Department:	INSTITUTE > Biotechnology Research Institute (BRI)
Depositing User:	DG MASNIAH AHMAD -
Date Deposited:	28 Apr 2025 15:52
Last Modified:	28 Apr 2025 15:52
URI:	https://eprints.ums.edu.my/id/eprint/43585

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