急性病毒性坏死病毒dUTPase基因的克隆、表达及其产物的酶学活性分析

根据已测定完成的栉孔扇贝急性病毒性坏死病毒(acute viral necrosis virus,AVNV)基因组序列,设计特异性引物,以提取的发病扇贝组织总DNA为模板,PCR扩增得到编码AVNV dUTPase的开放阅读框ORF074,将产物克隆至原核表达载体pET32a(+)中,构建表达质粒pET32a-dut。然后,将其转化至E.coli BL21(DE3)进行诱导表达。SDS-PAGE检测显示,诱导表达蛋白分子量约为46 ku,与预期表达蛋白大小一致。经Western-blotting及质谱分析鉴定,所表达蛋白即为重组dUTPase。表达产物经Co²⁺柱纯化后进行酶学活性测定。结果显示,重组dUTPase能特异性催化dUTP,EDTA可以抑制dUTPase的活性,而Mg²⁺可以增强其活性。

Cloning,expression of Chlamys farreri acute viral necrosis virus dUTPase gene and the determination of its enzymatic activity

Acute viral necrosis virus(AVNV)was reported as one causative agent responsible for summer mass mortality of adult Zhikong scallop(Chlamys farreri),which is widely cultured along northern China coast.To explore its pathogenesis at the molecular level,we cloned a gene which was predicted to encode AVNV dUTPase based on the genomic sequence of C.farreri AVNV completed by our laboratory.The gene encodes a protein of 248aa with a predicted molecular mass of 26.4 ku.To obtain the C.farreri AVNV Open Reading Frame 074,which probably encodes AVNV dUTPase,a pair of specific primers was designed based on the genomic sequence of C.farreri AVNV.Then this paper amplified the expected DNA by PCR,using the total genomic DNA extracted from infected C.farreri tissues as template.Amplified PCR fragments were subcloned into the prokaryotic expression vector pET-32a(+).After that,we transformed the recombinant plasmid pET32a-dut into E.coli BL21(DE3)strain and expressed it under IPTG induction.SDS-PAGE analysis showed that the molecular mass of the induced recombinant protein was about 46 ku.The Western-blotting and mass spectrometry analysis proved that the expressed protein is the target protein.Then we purified recombinant protein with Co²⁺ purification column and got the recombinant protein with a purity of more than 90%.The analysis of the enzymatic activity indicated that the recombinant dUTPase could specifically catalyse the hydrolysis of dUTP and the activity of enzyme was enhanced by Mg²⁺ while inhibited by EDTA.