siRNA vaccine against nerve necrosis virus in zebrafish
Date Issued
2006
Date
2006
Author(s)
Tsai, Han-Chuan
DOI
en-US
Abstract
Viral infections have been increasingly reported in cultured marine fish. Among these infections, viral encephalopathy and retinopathy (VER), caused by betanodaviruses (piscine nodavirus), is one of the most devastating, occurring in a variety of marine fish all over the world. Disease control would evolve an important issue for the whole world aquatic industry. Histological examination of tissues from the central nervous system and the retina often reveals areas of conspicuous tissue vacuolation and necrosis. Their genome consists of two molecules of messenger sense RNA. RNA1 is approximately 3.1 kb in size and carries the gene that encodes for an RNA-dependent RNA polymerase referred to as protein A; RNA2 is approximately 1.4 kb in size and contains the open reading frame that encodes the capsid protein.
Short interfering RNAs (siRNAs) have proved to be a useful tool in studying gene function. Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. In this thesis study, we want to development anti-fish virus by using RNAi technology, and nano-liposome was the delivery system that we used in this experiment.
We constructed 4 sets of siRNA vectors containing NNV's RNA2 gene in the position at 93, 585, 730 and 1024 nt. We also constructed the plasmids with HcRed gene regarding as siRNA detection marker. My experiment is separated into two parts: one is using siRNA expression vector inject zebrafish to single out stable line, and the F2 generation was challenge by NNV. There is higher survival rate compared to control. In the F1 generation, we could detect fluorescence marker gene in expression vector by RT-PCR. The other part：we used nano-liposome as DNA deliver tool with oral administration in zebrafish. Six different formulations of liposome were examined for the fluorescent marker gene of siRNA expression vector . The zebrafish was fed with nona-liposome, 4 h to 12 h after feeding, and the samples were collected at every interval of 2 h of this period. At 16 h after liposome feeding, there was strong fluorescence expressing in several organs including heart, eye and intestine etc. The result of in situ hybridization demonstrated vector-mediated siRNA could be expressed in zebrafishes. Therefore, vector-based siRNA delivered by nano-liposome has the potential to be the vaccine that against aquatic virus.in aquaculture.
Subjects
神經壞死病毒
核醣核酸干擾
斑馬魚
nerve necrosis virus
siRNA
zebrafish
Type
other
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