Clone And Expression Analysis Of Chlamys Farreri Acute Virus Necrosis Virus DUTPase Gene

Chinese scallop Chlamys farreri is one of the most important economic bivalvespecies widely farmed in the coastal provinces of north China. However, the outbreakof large scale mortality continuously lasted for several years starting from1997,which causes huge losses to the industry. Recently studies have confirmed that acutevirus necrosis virus （AVNV） is the main causative pathogen. The virus is adouble-stranded DNA virus, which is icosahedral with capsule envelope, and itsdiameter about130¹70nm.The genome of AVNV was completed in2009by Ren. Computer-assistedanalyses of the deduced amino acid sequences revealed that there are123potentialopen reading frames （ORF）, of which44putative gene products showed significanthomology to functionally characterized proteins of other species in the GeneBank/EMBL/GGBJ databases. These proteins included enzymes and structuralproteins involved in virus replication, nucleotide metabolism and modification andvirus-host interaction. ORF074was deduced to be the gene that encodes dUTPpyrophosphatase（dUTPase）, which mainly involves in DNA replication.dUTPase （deoxyuridine triphosphatase, dut; EC3.6.1.23） is a ubiquitous andimportant enzyme responsible for regulating levels of dUTP. it is closely related to DNA replication and virulence. To determine whether ORF074encodes functionalenzyme, and to figure out its role in the outbreak of AVND, we cloned the gene andconstruct its prokaryotic expression vector, expressed it in E.coli, then purified thereconstructed protein and analyzed its enzymatic activity. Secondly, constructed theeukaryotic expression vector pEGFP-N1-dut, expressed it in the grass carp kidneycells （CIK） and observed its subcellular localization in CIK. Addtionally, real-timePCR was conducted to analyze dut gene’s expression in C. farreri after infected byAVNV. Finally, to build AVNV vitro amplification system, providing technicalplatform for a more in-depth study of the infection mechanisms and pathogenesis, wecultured healthy scallops’ mantle, hemolymph, gills, adductor muscle, kidney in vitro,preliminary studied and established in vitro tissue culture technology of C. farreri,which provides a useful exploration for further study.1. Clone and prokaryotic expression of AVNV dUTPase gene. According toAVNV genome, a pair of specific primers was designed to amplify AVNV ORF074, a750bp band was obtained by1%agarose gel electrophoresis. Digested by restrictionenzyme, amplified PCR fragments were subcloned into the prokaryotic expressionvector pET-32a （+）. After that we transformed the recombinant plasmid pET32a-dutinto E.coli BL21（DE3） strain and expressed it under IPTG induction. SDS-PAGEanalysis showed that the molecular mass of the induced recombinant protein wasabout46kD. Then western-blot and mass spectrometry analysis proved that theexpressed protein is the target protein.2. Purification and enzymatic activity analyze of AVNV dUTPase. Therecombinant protein was purified using Co²⁺affinity chromatography. Then itsspecific activity, substrate specificity and influences on its activity by different metalions and EDTA was measured. The results was as follows:（1）. The recombinantdUTPase has a strong substrate specificity, which can specically catalytic dUTPhydrolysis.（2）. Mg²⁺、Ca²⁺、Co²⁺、Zn²⁺can enhance the catalytic activity of dUTPase,of which the effect of Mg²⁺is most significant and the order is Mg²⁺> Ca²⁺> Co²⁺>Zn²⁺.（3）. EDTA can inhibit the activity of the recombinant dUTPase, Mg²⁺、Ca²⁺、 Co²⁺、Zn²⁺can remove this inhibition to certain extent.3. Completed the transcriptional courses analyze of dut gene using real-time PCR.Total RNA was extracted from hepatopancreas of C. farreri at0,4,8,12,16,24,36,48h after being infected by AVNV. Then reverse transcription and fluorescentquantitative PCR was carried on to determine the gene expression courses.4. Determined dut gene’s transcriptional initiation site by5’RACE. Total RNA ofC. farreri’s hepatopancreas being infected16h afterward was extracted, reversetranscripted by random primer, and added poly（C） tail. Then basing on dUTPase genedesigned GSP primers and Adaptor （dG） was used to amplify, a250bp band was got,sequencing of which showed that the transcription start site was nucleotides T, whichwas10nts upstream of the initiation codon ATG.5. Recombinant eukaryotic expression vector pEGFP-N1-dut was constructedand transfected into the grass carp kidney cells （CIK）, then subcellular localizationwas analyzed after expressed. According to AVNV genome, a pair of specific primerswas designed. After digested by restriction enzyme, amplified PCR fragments weresubcloned into the eukaryotic expression vector pEGFP-N1. After that recombinantplasmid pEGFP-N1-dut was transformed into CIK cells and observe the expressionusing laser scanning confocal microscopy, the result of which shows that recombinantprotein expresses in both cytoplasm and nucleus of CIK cells.6. Five kinds of C. farreri tissue cells from adductor muscle, mantle, hemolymph,gill and kidney were cultured and subcultured. Cultured cells of the five tissues werepassed on to next generations. Results show that hemolymph cells die at the secondgeneration, the four other kinds of C. farreri tissue cells grow well till the thirdgeneration and die then.According to experimental results above, we can draw the following conclusions:AVNV ORF074could encode active dUTPase; the gene’s transcriptional initiationsite was nucleotide T, it could express in both cytoplasm and nucleus of CIK cells;The gene may be an early gene, which starts to express at4h postinfection, and16hpostinfection reaches the peak. In addition, we explored and established scallop tissueculture techniques preliminarily, accumulated some experience and lessons for establishment of AVNV proliferation in vitro.