Cloning, Expression And The Studies Of Functional Of Acute Viral Necrosis Virus Two Functional Genes

Acute viral necrosis virus(AVNV) was reported as the causativeagent responsible for summer mass mortality of adult Zhikongscallop(Chlamysfarreri)，which is widely cultured along northernChina coast．The genome of AVNV was completed by Ren.W.C.Computer-assisted analyses of the deduced amino acid sequencesrevealed that there are44putative gene products showed significanthomology tofunctionally characterized proteins in123potential openreading frames (ORF). Through gene cloning、 prokaryoticExpression、 Eukaryotic expresstion and the analysis of the functionof in vitro to understand the infection mechanism and the treatmentmechanism of AVNV that would provide effective prevention to provideAVNV infection way.The research contents and results are as follows:The first part:In this study, the open reading frame (ORF)86of AVNV wassuccessfully amplified based on the specific primers designedaccording to the complete genome sequences of AVNV. The geneencoded by ORF86in AVNVwas named as IAP-86in this study, sincethe homology of ORF86was firstly identified as encoding inhibitorof apoptosis protein (IAP)in baculovirus. We subcloned the amplifiedPCR fragments of IAP-86into the prokaryotic expression vectorpET32a(+), and obtained the recombinant plasmid pET32a-IAP86through thelinking of IAP-86gene to pET32a (+) plasmid．Then therecombinant plasmids were transformed into E.coil BL21(DE3) strainand expressed under the induction of IPTG．The SDS-PAGE analysisshowed that the molecular mass of the induced recombinant proteinwas about40ku．The expressed protein was verified through theWestern-blotting and mass spectrometry analysis．The second part：Then the recombinant protein was purified with Co2+purificationcolumn andmarked with FITC.We found IAP-86could combine withthe nuclear and the cytoplasm and inhibit the apoptosis of blood lymphocyte cell of C. farreri through coincubation of them. The cellapoptosis was inhibited by the recombinant of IAP-86according to theresult of apoptosis experiment, and the rate of apoptosis inhibition isabout7%.The third part:To understand the pathogenesis and the function of IAP-86protein of a strain of acute viral necrosis virus(AVNV) isolated fromAnadarabroughtonii,RNAwas extracted from the mantle ofmoribundA.Broughtoniithatinfectedwith AVNV, then， cDNA wasobtained by reverse transcription. Two pairs of nested reverse primerwere designed according to the ORF86sequence of AVNV completegenome sequence that registered in NCBI. Then the non-coding regionof5’and3’ end of the ORF86were amplified using the designedprimers by rapid amplification of cDNA ends (RACE) technique, andthe full-length cDNA sequencesspliced. Blast sequencealignmentillustrated that this gene hadthe homology to100%withoyster herpetovirus, and99%with the AVNV, moreover, overlappinggenes were found in the cDNA sequence. Bioinformatics analysisindicated that the protein did not contain either a signal peptide or atransmembrane region. The maximum hydrophobic index was1.800,minimum hydrophobic index was-3.456.Therewere eight potential phosphorylation sites(including five serine, two tyrosineandone threonine), a potential O-glycosylation sites and no potentialN-glycosylation sites. The epitope mainly focused on amino acids of8-11,14-16,28-39,75-76,88-95,97-100and147-158.The fourth part:In the based of failure that use prokaryotic expression techniqueexpressed AVNV SF2helicase,we tried to use yeast expresstion systemto AVNV SF2helicase gene proceed eukaryotic expresstion. This partwe subcloned the amplified PCR fragments of ORF-66into theeukaryotic expression vector pPIC9K, and obtained the recombinantplasmid pPIC9K-ORF66through the linking of ORF-66gene topPIC9K plasmid．...