Gene Analysis And Eukaryotic Expression Of Capsid Protein Gene From Fish Viral Nervous Necrosis Virus

Betanodavirus infection is responsible for Viral nervous necrosis (VNN) an emerging disease, which was described for the first time at the end of the 1980's and is now prevalent in all continents except Africa. The infection of Betanodavirus has caused great economic losses of mariculture in many countries. Betanodavirus are RNA viruses whose genome consists of two molecules: RNA1 encoding for the RNA-dependent RNA-polymerase and RNA2 encoding for the coat protein. According to phylogenetic analysis of the coat protein gene. The infection of Betanodavirus also caused great loss inSouthern China, especially in the larvae of cultured marine fish.Six nervous necrosis virus were isolated from cultured yellow grouper in 2006 in shore of china.Total viral RNA genome was extracted.Based on the complete sequences of RNA2 which have been submitted to Genbank,a pair of primers were designed and the coat protein genes from the 6 isolates were amplified by means of reverse-transcriptase polymerase chain reaction(RT-PCR).The RT-PCR products were ligated into the PMD18-T vector and transformed into E.coli DH5a.The recombinant plasmid were sequenced respectively.There was more than 97.3% nucleotide identityies and 79.6-99.1% among the deduced amino acid sequences within the six isolates. Phylogentic analysis showed that the isolates shared evolutionary direction with EVNNV,ETNNV and MNNV .It was revealed that these isolates belonged to genotype RGNNV. It was deduced that the strains of RGNNV genotype were dominant and prevalent in spotted grouper cultured in China during the past several years.The cp gene of NNV 0603 Strain was amplified successfully by reverse-transcription polymerase chain reaction (RT-PCR), and inserted into plasmid pFastBacI of the Baculovirus expression system Bac-To-Bac to form plasmid pFastBac-cp. Through homologous recombination between pFastBac-cp and bacmid which is genome modified of Autogragha California nuclear polyhedrosis virus (AcNPV), recombinant shuttle vector named Bacmid-cp was obtained in E. coli DH₁₀Bac. After tranfection with Cellfectin Reagent, recombined Baculovirus (AcNPV-cp) was developed in Sf9 cells. The specific 37kD band was showed by analysis of SDS-PAGE and Western blotting, which indicated that NNV cp protein had expressed in insect cells Sf9. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in cp crude extract and in the infected Sf9 cells by electron microscope. The VLPs were similar to NNV whole virion, and became a crystalline structure in cytoplasm.