Cloning,Expression Of Chlamys Farreri Acute Viral Necrosis Virus Primase And The Determination Of Its Functions

The scallop Chlamys farreri has been performed more than20years and its culture in commercial scale which is one of the major species cultured in North China. It brings considerable economic benefits to the coastal areas of China’s Shandong and Liaodong Peninsula, However, since1990, The great expansion and intensification have induced the occurrence of disease and mass death of adult and seeding of Chlamys farreri (mortality rate in more than90%) in north coast of China. Which has been becoming the major limiting factor in the development of the scallop industry, More seriously, striking the economic process in the north coastal region of China. Recently studies have confirmed that acute viral necrosis virus (AVNV) is the main causative pathogen about the Chlamys farreri outbreak of large scale mortality continuously. The virus is a double-stranded DNA virus and take the sphere, The virions could be found in the connective tissue and interstitial cells of digestive gland, kidney, mantle, and intestine. The genome of AVNV was completed by Ren.W.C. Computer-assisted analyses of the deduced amino acid sequences revealed that there are44putative gene products showed significant homology tofunctionally characterized proteins in123potential open reading frames (ORF). These proteins included enzymes and structural proteins involved in virus replication, nucleotide modification and metabolism and virus-host interaction. ORF024was deduced to be the gene that encodes primase, which mainly involves in DNA replication. The primase is the RNA polymerase which exist in organism and the virus is closely related to DNA replication and viral activity. To determine whether ORF024encodes specific functional primase, and to figure out its role in the Infection, replication in AVNV and outbreak of AVND, we designed the specific primers of AVNV primase’s gene and cloned the gene ORF024, and construct its prokaryotic expression vector expressed it in E.coli, and then purified the reconstructed protein and analyzed its enzymatic activity. Secondly, Making use of natural green tea extract (tea polyphenols, TP) inhibit AVNV primase to explore TP’s antiviral mechanism. In addition, By real-time PCR conducted to analyze primase gene’s expression in C. farreri(after infected by AVNV and infected by AVNV with TP) in different tissues and different times to analysis transcription and timing expression in mRNA level to explore the gene of AVNV primase, and explore TP wheather could inhibit AVNV replication in the scallop, the Inhibition rate cuold be used to theoretical basis for TP anti-AVNV mechanism.1. Clone and prokaryotic expression of AVNV primase gene.In the light of AVNV genome, a pair of specific primers was designed to amplify AVNV ORF024, a1050bp band was obtained by1%agarose gel electrophoresis, By restriction enzyme’s double digestion, The amplified PCR fragments were subcloned into the prokaryotic expression vector pET-32a (+). After that, the recombinant plasmid pET32a-prim was transformed into E.coli BL21(DE3) strain and expressed it under IPTG (lmmol/L) induction about4-5h. SDS-PAGE analysis showed that the molecular mass of the induced recombinant protein was about60KDa. By western-blot and mass spectrometry analysis proved that the expressed protein is the recombinant primase.2. Purification and enzymatic activity analyze of AVNV primase.Using Co2+affinity chromatography purified the recombinant protein. Then its specific activity, substrate specificity and influences on its activity by different metal ions and EDTA was measured. The results as follows:(1) Recombinant primase have some catalytic activity in synthesis of random primers.(2) With the single-stranded template of poly (d C), the recombinant primase has a strong substrate specificity, which can specically catalytic GTP hydrolysis.(3)Zn2+, Na+,K+,Mg2+can enhance the catalytic activity of primase, The Zn2+(0.1mmol/L)’s promotion rate was5%, The Na+(5mmol/L)’s promotion ratio was only3%, and Mn2+have significantly inhibited the catalytic activity of the recombinant primase.(4) EDTA also have significantly inhibited the catalytic activity of the recombinant primase, the higher concentration, the stronger the inhibition rate, EDTA (10mmol/L)’s inhibition rate is up to12.20%. 3. Using different concentrations of TP solution which was dissolved in the reaction system to explore the effects of TP on activity of recombinant primase in vitro and primer synthesis, under the concentration of7.81μg/ml,31.25μg/ml and125μg/ml of TP, with the concentration of TP’s increases, Primers activity inhibition rate gradually increases, and the amount of primer synthesis gradually increases.4. Completed the transcriptional courses analyze of primase gene using real-time PCR. Total RNA was extracted from mantle and gills of C. farreri at0,4,8,16,24,36,48and72h after being infected by AVNV and AVNV with TP. Then reverse transcription and fluorescent quantitative PCR was carried on to determine the primase gene expression courses to determine significant differences in the two experimental groups. AVNV primase gene expression in the mantle and gills respectively reached the maximum at16and8h; TP (7.81μg/ml) could inhibit the replication of AVNV (in the mantle andthe gills), and its inhibition rates were5and10-folds.According to experimental results above, we can draw the following conclusions: AVNV ORF024could encode active primase; different metal ions have significant effect on the primase activity, such as Zn2+and Na+play a strongly promotion catalytic role in AVNV primase, at the same time, Mn2+and EDTA play a strongly inhibition catalytic role in AVNV primase. The primase gene is an early gene, there is different maximum expression level and different time of AVNV primase have significant differences in mantle and gills of C. farreri,8h postinfection reaches the peak in gills. However,16h postinfection reaches the peak in mantle. In addition, primase gene expression from infected C. farreri AVNV mixed TP is significantly lower from infected C. farreri AVNV, described above, the TP has a certain inhibition role in AVNV invasion to C. farreri.