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Expression and enzymatic activity analysis of primase from acute viral necrosis virus

Acute viral necrosis virus(AVNV)was reported as one causative agent responsible for mass mortality of adult Chlamys farreri,which is widely cultured along northern China coast.To explore its pathogenesis at the molecular level,a gene was cloned which was predicted to encode AVNV primase based on the genomic sequence of AVNV completed by our laboratory.The gene encodes a protein of 350 aa with a predicted molecular mass of 60 ku.To obtain AVNV ORF 024,which probably encodes AVNV primase,a pair of specific primers was designed based on the genomic sequence of AVNV.Then this paper amplified the expected DNA by PCR,and used the total genomic DNA extracted from infected C.farreri tissues as template.Amplified PCR fragments were cloned into the prokaryotic expression vector pET-32a(+).After that,the plasmid pET32a-prim was transformed into E.coli BL21(DE3)stain,and AVNV primase was expressed under IPTG induction.SDS-PAGE analysis showed that the two induced recombinant proteins' molecular mass was about 60 and 55 ku,the Western-blotting and mass spectrometry analysis proved that the expressed protein(60 ku)was the primase,while another expressed protein(55 ku)had some of the primase peptide fragments.Meanwhile,the experiments showed that Pico-Green(it was able to specifically bind to a random short segments of RNA)as the fluorescent dye which had a stable fluorescence intensity in 30 min.So the 30 min was the termination time for the enzymatic activity analysis of the recombined primase.In the presence of template of the poly(d C),the analysis of the enzymatic activity indicated that the recombinant primase could specifically catalyze the hydrolysis of GTP.The enzymatic activity of primase was enhanced by 0.1 mmol/L Zn2+ but inhibited by 1 mmol/L Mn2+ and EDTA.